The endogenous opioid enkephalins (ENK) are highly expressed in the central nucleus of the amygdaloid complex (CeA) where several lines of evidence point to a potential role in the modulation of fear and anxiety. In this study, we aimed to assess the role of CeA ENK using local injections of a lentiviral vector expressing a short hairpin RNA (shRNA) targeting ENK in Sprague–Dawley rats. We injected this vector in the CeA and a 56% downregulation of ENK mRNA was observed in animals when compared with scrambled shRNA animals. Anxiety-like behaviors were also assessed using the elevated plus maze and social interaction test. There was an increase in exploration of open arms of the elevated plus maze in ENK knockdown animals compared with controls, but no change in social interaction. In addition, we used the contextual fear conditioning procedure to assess fear expression and learning in these animals. There was a reduction in freezing induced by acute shocks during the training procedure. Interestingly, associative learning was not affected, and ENK knockdown animals displayed an equivalent freezing when re-exposed to the conditioning chamber 48 h later. These results contrast with knockout mice studies, which ascribed anxiolytic properties to ENK, and they demonstrate the need for a thorough understanding and characterization of neuroanatomically distinct ENK pathways.