To control viral infection, vertebrates rely on both inducible interferon responses and less well-characterized cell-intrinsic responses composed of “at the ready” antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell-intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7 but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor. Display omitted •cDNA screen reveals E3 ligase TRIM7 as a potent inhibitor of human enteroviruses•TRIM7 targets viral 2BC protein for ubiquitination and degradation•A single T323A amino acid change in viral 2C protein confers resistance to TRIM7•TRIM7-resistant viral variant replicates robustly in mice and causes severe disease The E3 ubiquitin ligase TRIM7 is an antiviral effector that restricts multiple human enteroviruses.