Recent studies have revealed nesfatin-1 as a hypothalamic neuropeptide, regulating food intake, energy expenditure and reproduction primarily by acting on the hypothalamic-pituitary-gonadal axis. Nesfatin-1 is also localized in several peripheral tissues including testes. However, functional significance of nesfatin-1 in testicular activities is not yet well documented in mammals. Therefore, this study was aimed to elucidate the direct effects of nesfatin-1 on testicular markers for steroid productions, spermatogenesis, metabolic changes and oxidative stress. The results revealed the expression of both protein and mRNA of nesfatin-1 in the testes of adult mice. The testes treated in vitro with nesfatin-1 showed significant increase in testosterone production, which correlated significantly with increased expression of steroidogenic markers and insulin receptor proteins in the testes. Furthermore, the in vitro treatment with nesfatin-1 showed stimulatory effects on spermatogenesis by promoting cell proliferation (PCNA) and survival (Bcl2), while inhibiting apoptosis (caspase-3) in the testes. The nesfatin-1 treatment in vitro further increased the expression of insulin receptor and GLUT8 proteins, in parallel with increase in the intra-testicular transport of glucose and production of lactate. This nesfatin-1 induced enhanced transport of energy substrate (glucose and lactate) may be responsible for promoting spermatogenesis and steroidogenesis. Nesfatin-1 significantly reduced oxidative stress and nitric oxide, which may also be responsible for stimulatory effects on testicular activities. The present finding suggests that nesfatin-1 acts via paracrine manner to increase sperm count and fertility, thus promoting the testicular function.