Agnihotra (IAST: Agnihotra, Devnagari: अग्निहोत्र) refers to the yajna of offering ghee into the sacred fire as per strict rites, and may include twice-daily heated milk offering made by those in the Śrauta tradition. The ritual has been described by P.E. Dumont as a "fertility charm", and as a "solar charm" which symbolically preserved and created the sun at nightfall and sunrise.

This tradition dates back to the Vedic age; the Brahmans perform the Agnihotra ritual chanting the verses from the Rigveda. It is part of a pan-Indo-Iranian heritage, which includes the related Iranian fire-worship ritual called Zoroastrian Yasna Haptaŋhāiti ritual mentioned in the Old Avestan. In the historical Vedic religion, Agnihotra was the simplest public rite, and the head of every Brahmin and Vaishya family was required to conduct it twice daily. It was already popular in India with Upaniṣads as religious performance. The tradition is now practiced in many parts of South Asia in the Indian sub-continent, including primarily India and also in Nepal. The Brahmin who performs the Agnihotra ritual is called an Agnihotri.

The ritual is conducted twice daily, right before or after sunrise and after sunset or the appearance of the first night star. The morning and evening agnihotras differ by the mantras and chants made by the officiants. At least four people take part in the sacrifice: the sacrificer, who hires priests to perform the ceremony (Brahmin), his wife, an Adhvaryu (Sanskrit: अध्वर्यु) and a milker.

Vedic rituals are typically performed by four priests: the aforementioned adhvaryu, who is responsible for the physical details of the sacrifice and chants the Yajurveda, a hotṛ (Sanskrit: होतृ) who recites the Rigveda, an udgātṛ (Sanskrit: उद्गातृ) who sings hymns of the Samaveda, and a brahman (Sanskrit: ब्राह्मण, romanized: brāhmaṇa) who supervises the ceremony, and recites the Atharvaveda while correcting any errors that may occur. There are three fires: an eastern offertorial fire called an āhavanīya (Sanskrit: आहवनीय) lit in a square fire pit, a western fire called the gārhapatya (Sanskrit: गार्हपत्य) lit in a circular fire pit, which represents the householder's fire, and a southern fire simply called the dakṣiṇāgni (Southern fire) (Sanskrit: दक्षिणाग्नि). During the ceremonies, a ritual poker, pot (Sanskrit: अग्निहोत्रस्थाली, romanized: agnihotrasthālī), a spoon (Sanskrit: स्रुव, romanized: sruva), and ladle (Sanskrit: अग्निहोत्रहवनी, romanized: agnihotrahavanī) are all used. At the centre of the ritual space is an earthen altar (Sanskrit: वेदी, romanized: vedī) where the tools to perform the ritual are placed.

The most common offering in the Agnihotra ritual is milk. The Kātyāyana Śrautasūtra advises devotees to sacrifice with milk to obtain cattle or a position in heaven. Alternative offerings for different goals are also recommended: the acquisition of a village with rice gruel, strength with plain rice grains, refined senses with curds, and "sharpness" or "spirit" (Sanskrit: तेज, romanized: teja) with ghee. Performance of the Agnihotra as a purely ritual obligation is known as nitya (Sanskrit: नित्य), while performance with a special purpose is called kāmya. (Sanskrit: नित्य)

When the sacrificial area has been cleaned and the sacrificial fire lit, a cow is brought to the grounds and the milker, an ā́rya and not a śūdra, recites mantras before it, then brings the calf to the right side of its mother before beginning the milking. The milk is kept in the agnihotrasthālī, which can also only be made by an ā́rya.

When the milking is complete, the adhvaryu pours water around the three fires, before boiling the collected milk on coals collected from the gārhapatya. The adhvaryu draws milk into the ladle, pouring it first onto the āhavanῑya twice: first while reciting mantras, and the second silently as an offering to Prajapati. The mantra for offering the milk during the evening is "Agni is the light. The light is Agni, svaha". (Sanskrit: अग्निर् ज्योतिर् ज्योतिर् अग्निः स्वाहा, romanized: agnir jyotir jyotir agniḥ svāhā). In the morning, Surya is substituted for Agni: (Sanskrit: सूर्यो ज्योतिः ज्योतिः सूर्यः स्वाहा, romanized: sūryo jyotiḥ jyotiḥ sūryaḥ svāhā).

When the libations are complete, the ladle is cleaned with Darbha grass and refilled with water. It is then heated on the āhavanīya as additional mantras are recited, and poured onto the altar as an additional libation. In certain versions of the ritual (but not that contained in the Tattirīya Brahmana), this is followed by a blade of grass being offered to the āhavanīya. When the ceremony is complete, the adhvaryu sips some of the leftover water, recites the mantra "From Rta I have found Satya" and pours water on his head.