Gossypol is a kind of yellow polyphenolic compounds extracted from root stem and seed of the cotton plant. In the present study, we investigated its immunosuppressive mechanism by using BALB/c mouse T lymphocytes in vitro. When mouse splenocytes was incubated with gossypol, the extract effectively suppress the overproduction of the cell stimulated by concanavalin A (ConA) in a dose manner. This inhibitive activity was mainly due to interfering Th1 and Th2 cytokines production and decreasing CD4(+) T cell populations and ratio of CD4(+)/CD8(+). Furthermore, we also showed that signal transduction via NF-κB, NFAT and AP-1 are critical to the ConA-induced T cell activation in mice. The data revealed that gossypol could down-regulate activation of ConA-induced NF-κB, NFAT and AP-1 signal transduction pathways in mouse T lymphocyte. These observations indicated that gossypol exhibited immunosuppressive effects by inhibition T lymphocyte activation in vitro.