In the current study, we tested a hypothesis that CD36 fatty acid (FA) transporter might affect insulin sensitivity by indirect effects on FA composition of adipose tissue. We examined the effects of CD36 downregulation by RNA interference in 3T3-L1 adipocytes on FA transport and composition and on sensitivity to insulin action. Transfected 3T3-L1 adipocytes, without detectable CD36 protein, showed reduced neutral lipid levels and significant differences in FA composition when levels of essential FA and their metabolites were lower or could not be detected including gamma linolenic (C18: 3 n6), eicosadienic (C20: 2 n6), dihomo-gamma linolenic (C20: 3 n6), eicosapentaenoic (EPA)(C20: 5 n3), docosapentaenoic (DPA)(C22: 5 n3), and docosahexaenoic (DHA)(C22: 6 n3) FA. Transfected 3T3-L1 adipocytes exhibited a significantly higher n6/n3 FA ratio, reduced∆ 5-desaturase and higher∆ 9-desaturase activities. These lipid profiles were associated with a significantly reduced insulin-stimulated glucose uptake (4.02±0.1 vs. 8.42±0.26 pmol. 10-3 cells, P= 0.001). These findings provide evidence that CD36 regulates FA composition thereby affecting sensitivity to insulin action in 3T3-L1 adipocytes.