Serine phosphorylation of IRS-1 and the induction of suppressor of cytokine signaling 3 (SOCS3) is recently well documented as the mechanisms for the insulin resistance. However, the relationship between these two mechanisms is not fully understood. In this study, we investigated the involvement of SOCS3 and IRS-1 serine phosphorylation in TNF-induced insulin resistance in 3T3-L1 adipocytes. TNF transiently stimulated serine phosphorylation of IRS-1 from 10 min to 1 h, whereas insulin-stimulated IRS-1 tyrosine phosphorylation was inhibited only after TNF treatment longer than 4h. These results suggest that serine phosphorylation of IRS-1 alone is not the major mechanism for the inhibited insulin signaling by TNF. TNF stimulation longer than 4 h enhanced the expression of SOCS3 and STAT3 phosphorylation, concomitantly with the production of IL-6. Anti-IL-6 neutralizing antibody ameliorated suppressed insulin signaling by 24 h TNF treatment, when it partially decreased SOCS3 induction and STAT3 phosphorylation. These results suggest that SOCS3 induction is involved in inhibited insulin signaling by TNF. However, low level expression of SOCS3 by IL-6 or adenovirus vector did not affect insulin-stimulated IRS-1 tyrosine phosphorylation. Interestingly, when IRS-1 serine phosphorylation was enhanced by TNF or anisomycin in the presence of low level SOCS3, IRS-1 degradation was remarkably enhanced. Taken together, both IRS-1serine phosphorylation and SOCS3 induction are necessary but one of the pair is not sufficient for the inhibited insulin signaling. Chronic TNF may inhibit insulin signaling effectively, because it causes both IRS-1 serine phosphorylation and the following SOCS3 induction in 3T3-L1 adipocytes.