Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses.