The present study used in vivo and in vitro methods to characterize the effects of MeHg on key neuroendocrine biochemicals. First, adult female yellow perch (Perca flavescens) were exposed to dietary MeHg (0, 0.5, 5, 50 ppm) for 16 weeks throughout the gonadal development period. In different brain regions and liver, GABA-benzodiazepine (BZ), dopamine (D2), estrogen (ERs) and androgen receptors (ARs), and monoamine oxidase (MAO) were measured. Saturation curves revealed a MeHg-dependent increase of the maximum binding (Bmax) of D2 receptor in the hypothalamus versus controls (284–606%), concomitant with a decrease in receptor affinity (up to 984% lower). The activity of MAO was enhanced in different brain regions of exposed fish (240–383% in the hypothalamus). MeHg concentrations were negatively correlated with the number of ERs in all brain regions except for the hypothalamus. For the 5 and 50 ppm exposure groups, MeHg concentrations were positively correlated with the number of ARs (130–329% and 225% for brain regions and liver, respectively compared to controls). In vitro exposures were conducted in parallel using different fish species (giant danio, goldfish, yellow perch, lake trout) to test the inhibitory potential of MeHgCl or HgCl2 (0.01–320 μM). There was no evidence of impacts on receptors, but the activities of GS, GAD and MAO (except in yellow perch) were inhibited by both MeHgCl and HgCl2 in a species-dependent manner. In general, these findings show that environmentally relevant dietary exposures to MeHg can affect key neuroendocrine receptors and enzymes important to fish reproduction.