Humans and many animals exhibit freezing behavior in response to threatening stimuli. In humans, inappropriate threat responses are fundamental characteristics of several mental illnesses. To identify small molecules that modulate threat responses, we developed a high-throughput behavioral assay in zebrafish (Danio rerio) and characterized the effects of 10,000 compounds on freezing behavior. We found three classes of compounds that switch the threat response from freezing to escape-like behavior. We then screened these for binding activity across 45 candidate targets. Using target profile clustering we implicated the sigma-1 receptor in the mechanism of behavioral switching and confirmed that known sigma-1 ligands also disrupt freezing behavior. Furthermore, mutation of the sigma-1 gene prevented the behavioral effect of escape-inducing compounds. The compound ‘finazine’ potently bound mammalian sigma-1 and altered rodent threat response behavior. Thus, pharmacological and genetic interrogation of the freezing response revealed sigma-1 as a mediator of vertebrate threat responses.