Estrogen is believed to be involved in regulation of the differentiation, survival, or function of diverse immune cells as well as in many autoimmune and inflammatory diseases. However, the mechanisms behind the immunomodulatory effects of estrogen are poorly understood. Previously, we have shown that natural estrogen can upregulate IFN-γ and IFN-γ-mediated-inflammatory events (iNOS, nitric oxide, COX-2). Since IFN-γ is regulated by T-bet, in this study, we investigated whether estrogen induces T-bet expression in primary murine splenocytes. We found that in vivo estrogen treatment primes splenocytes for early upregulation of T-bet upon activation by T cell stimulants, Concanavalin-A (Con-A) or anti-CD3 antibodies. The expression of T-bet protein was not altered by IL-12 while IFN-γ had partial effects on T-bet in splenocytes from estrogen-treated mice. Notably, T-bet expression increased in Con-A-activated splenocytes from estrogen-treated mice in the presence of IL-27. Together, our studies show that in vivo estrogen exposure primes lymphocytes towards Th1 type development by promoting/upregulating T-bet expression, which is upregulated in part by IFN-γ and IL-27. Given that T-bet is a potent inducer of IFN-γ, these studies may lead to new lines of investigation in relation to many female-predominant autoimmune diseases and inflammatory disorders.