HindIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg²⁺ via hydrolysis.

The cleavage of this sequence between the AA's results in 5' overhangs on the DNA called sticky ends:

5'-A |A G C T T-3'

3'-T T C G A| A-5'

Restriction endonucleases are used as defense mechanisms in prokaryotic organisms in the restriction modification system. Their primary function is to protect the host genome against invasion by foreign DNA, primarily bacteriophage DNA. There is also evidence that suggests the restriction enzymes may act alongside modification enzymes as selfish elements, or may be involved in genetic recombination and transposition.

The structure of HindIII is complex, and consists of a homodimer. Like other type II restriction endonucleases, it is believed to contain a common structural core comprising four β-sheets and a single α-helix. Each subunit contains 300 amino acids and the predicted molecular mass is 34,950 Da. Despite the importance of this enzyme in molecular biology and DNA technology, little information is available concerning the mechanism of DNA recognition and phosphodiester bond cleavage. However, it is believed that HindIII utilizes a common mechanism of recognition and catalysis of DNA found in other type II enzymes such as EcoRI, BamHI, and BglII. These enzymes contain the amino acid sequence motif PD-(D/E)XK to coordinate Mg²⁺, a cation required to cleave DNA in most type II restriction endonucleases. The cofactor Mg²⁺ is believed to bind water molecules and carry them to the catalytic sites of the enzymes, among other cations. Unlike most documented type II restriction endonucleases, HindIII is unique in that it has little to no catalytic activity when Mg²⁺ is substituted for other cofactors, such as Mn²⁺.

Despite the uncertainty concerning the structure-catalysis relationship of type II endonucleases, site-directed mutagenesis of the restriction endonuclease HindIII has provided much insight into the key amino acid residues involved. In particular, substitutions of Asn for Lys at residue 125 and Leu for Asp at residue 108 significantly decreased DNA binding and the catalytic function of HindIII. In a separate mutagenesis study it was shown that a mutation at residue 123 from Asp to Asn reduced enzymatic activity. Despite the fact that this residue is most likely responsible for the unwinding of DNA and coordination to water rather than direct interaction with the attacking nucleophile, its specific function is unknown.

While restriction enzymes cleave at specific DNA sequences, they are first required to bind non-specifically with the DNA backbone before localizing to the restriction site. On average, the restriction enzyme will form 15-20 hydrogen bonds with the bases of the recognition sequence. With the aid of other van der Waals interactions, this bonding facilitates a conformational change of the DNA-enzyme complex which leads to the activation of catalytic centers.