Recent research indicates that men may have evolved mechanisms dedicated to detecting and responding to the risk of partner infidelity. Because activation of these “anti-cuckoldry” mechanisms depends on partner infidelity, or the perception of partner infidelity, existing evidence for such mechanisms relies on correlational data. The current study tests several predictions regarding men’s anti-cuckoldry mechanisms in an experimental design. As predicted, the results demonstrated: (1a) experimental activation of men’s anti-cuckoldry mechanisms by presenting them with a vignette depicting a female partner’s sexual infidelity; (1b) no activation of men’s anti-cuckoldry mechanisms by presenting them with a vignette depicting a sexual encounter without female infidelity; (2) experimental activation of men’s anti-cuckoldry mechanisms was influenced by their perceived risk of partner infidelity; and (3) women were not influenced by the partner infidelity manipulation.