Estrogen regulation of inflammatory responses has broad physiological and pathological consequences. However, the molecular mechanism of estrogen regulation of inflammation is still poorly understood. In this study, we report that activation of both STAT-1 and NF-κB signaling is essential for Con A-induced inducible NO synthase (iNOS) and NO in murine splenocytes. Estrogen enhances STAT-1 DNA-binding activity without increasing the expression of phosphorylated and total STAT-1 protein. We have recently reported that estrogen blocks the nuclear expression of NF-κB p65 and modifies nuclear NF-κBp50. Here, we demonstrated that both nuclear STAT-1 and NF-κB are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-κB activity/signaling in splenocytes from estrogen-treated mice. Inhibition of serine protease activity with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) restores the nuclear expression of full-length STAT-1 and NF-κB proteins, and resulted in decreased STAT-1 DNA-binding activity and formation of NF-κB p65/p50 binding complexes in nuclei of splenocytes from estrogen-treated mice. Consequently, there is significantly decreased iNOS and IFN-γ production in AEBSF-treated splenocytes from estrogen-treated mice, which suggests a positive regulatory role of truncated STAT-1 and/or NF-κB. Interestingly, there is increased production of MCP-1 in STAT-1 or NF-κB small interfering RNA-transfected cells, as well as in AEBSF-treated splenocytes from estrogen-treated mice. These data suggest a differential role of truncated STAT-1 and NF-κB in regulation of various inflammatory molecules in splenocytes from estrogen-treated mice. Together, our data reveal a novel molecular mechanism of estrogen-mediated promotion of inflammatory responses, which involves posttranslational modification of STAT-1 and NF-κB proteins.