Thalassospiramides comprise a large family of lipopeptide natural products produced by Thalassospira and Tistrella marine bacteria. Here we provide further evidence of their nanomolar inhibitory activity against the human calpain 1 protease. Analysis of structure-activity relationship data supported our hypothesis that the rigid 12-membered ring containing an α,β-unsaturated carbonyl moiety is the pharmacologically active functional group, in contrast to classic electrophilic "warheads" in known calpain inhibitors. Using a combination of chemical modifications, mass spectrometric techniques, site-directed mutagenesis, and molecular modeling, we show the covalent binding of thalassospiramide's α,β-unsaturated carbonyl moiety to the thiol group of calpain's catalytic Cys115 residue by a Michael 1,4-addition reaction. As nanomolar calpain inhibitors with promising selectivity and low toxicity from natural sources are rare, we consider thalassospiramides as promising drug leads.