Suppressor of cytokine signaling (SOCS)-3 can act as a regulator of energy metabolism and cytokine signaling in fat cells. It is regulated by hormones and toxicological factors. However, the action of cycloheximide on expression of adipocyte SOCS-3 gene is unknown. Using 3T3-L1 adipocytes, we found that cycloheximide up-regulated SOCS-3 mRNA expression in dose- and time-dependent manners. Treatment with actinomycin D prevented cycloheximide-stimulated SOCS-3 mRNA expression, suggesting that the effect of cycloheximide requires new mRNA synthesis. While cycloheximide was shown to increase activities of MEK1 and JNK, signaling was demonstrated to be inhibited by pretreatment with either MEK1 inhibitors U0126 and PD98059, or with the JNK inhibitor SP600125. U0126 and PD98059, respectively, reduced cycloheximide-stimulated SOCS-3 mRNA expression, but SP600125 did not antagonize cycloheximide effect. Moreover, cycloheximide was observed to up-regulate expression of other SOCS family members, such as SOCS-1, -2, -4, -5, -6, -7, and cytokine-inducible SH2-containing protein (CIS)-1 mRNAs. Such effects varied with the dosage and duration of cycloheximide treatment. These results imply the functional MEK1/ERK-mediated pathway is necessary for the cycloheximide stimulation of SOCS-3 gene expression.