Cefalotin (INN) /ˌsɛfəˈloʊtɪn/ or cephalothin (USAN) /ˌsɛfəˈloʊθɪn/ is a first-generation cephalosporin antibiotic with broad spectrum antibiotic activity. It was the first cephalosporin marketed (1964) and continues to be widely used. Cefalotin is used for bacterial infections of the respiratory tract, urinary tract, skin, soft tissues, bones and joints, sepsis, peritonitis, osteomyelitis, mastitis, infected wounds, and post-operational infections.

It is an intravenously administered agent with a similar antimicrobial spectrum to cefazolin and the oral agents cefalexin and cefadroxil. Cefalotin sodium is marketed as Keflin (Lilly) and under other trade names.

The compound is a derivative of thiophene-2-acetic acid.