Estradiol (17β-estradiol, E2) is a crucial estrogen hormone that regulates sexual, cognitive, social and affective behaviors in various species. However, complex central nervous system (CNS) effects of E2, including its activity in males, remain poorly understood. The zebrafish (Danio rerio) is rapidly becoming a powerful novel model system in translational neuroscience research. Here, we evaluate the effects of a single 24-h exposure to 20 μg/L of E2 on behavioral and endocrine (cortisol) responses in adult male zebrafish. Overall, E2 exerted pro-social effect in the social preference test, reduced whole-body cortisol levels, elevated exploration in the novel tank test and increased the shoal size in the shoaling test, indicative of an anxiolytic-like profile of this hormone in male zebrafish. Supporting mounting human and rodent evidence on the role of E2 in behavioral regulation, the observed pro-social and anxiolytic-like effects of E2 in male zebrafish reinforce the use of this aquatic organism in studying steroid-mediated CNS mechanisms of complex affective and social behaviors.