FGIN-1-27 is an agonist at the translocator protein 18 kDa (TSPO), a cholesterol transporter that is associated with neurosteroidogenesis. This protein has been identified as a peripheral binding site for benzodiazepines; in anamniotes, however, a second TSPO isoform that is absent in amniotes has been implicated in erythropoiesis. Functional conservation of the central benzodiazepine-binding site located in the GABAA receptors has been demonstrated in anamniotes and amniotes alike; however, it was not previously demonstrated for TSPO. The present investigation explored the behavioral effects of FGIN-1-27 on an anxiety test in zebrafish (Danio rerio, Family: Cyprinide) and on a mixed anxiety/panic test on wall lizards (Tropidurus oreadicus, Family: Tropiduridae). Results showed that FGIN-1-27 reduced anxiety-like behavior in the zebrafish light/dark preference test similar to diazepam, but with fewer sedative effects. Similarly, FGIN-1-27 also reduced anxiety- and fear-like behaviors in the defense test battery in wall lizards, again producing fewer sedative-like effects than diazepam; the benzodiazepine was also unable to reduce fear-like behaviors in this species. These results A) underline the functional conservation of TSPO in defensive behavior in anamniotes; B) strengthen the proposal of using anamniote behavior as models in behavioral pharmacology; and C) suggest TSPO/neurosteroidogenesis as a target in treating anxiety disorders.