Class II major histocompatibility complex (MHC) proteins are important for specific recognition of foreign antigens by the immune system. Previously we showed that 17β-estradiol (E2) down-regulates class II MHC expression by attenuation of histone acetylation and cAMP response element binding protein (CREB)-binding protein recruitment to the class II MHC promoter. Estrogen signals through nuclear receptors to mediate genomic effects; however, estrogen is also known to mediate rapid nongenomic effects. Our observation that ER antagonists fail to prevent E2 inhibition of class II MHC expression suggests that E2 is signaling in a neoclassical manner. We find that E2, as well as the antiestrogens tamoxifen (TAM) and ICI 182,780 (ICI), inhibit class II MHC expression through activation of the c-Jun N-terminal kinase (JNK) pathway. Pharmacological JNK inhibitors reverse the inhibitory effects of E2, TAM, and ICI on class II MHC expression. E2, TAM, and ICI activate the JNK pathway and subsequently activate c-Jun and activating transcription factor-2 transcription factors. Our results demonstrate that blocking E2 activation of the JNK signaling pathway prevents estrogen-mediated attenuation of histone acetylation and CREB-binding protein recruitment to the class II MHC promoter. Collectively, these findings demonstrate that the JNK signaling pathway is necessary for E 2-mediated inhibition of class II MHC expression.