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.