To investigate the effects of testosterone (T) on inflammatory cytokines (IL-6, MCP-1) production, insulin sensitivity of adipocyte and changes of macrophage phenotypes in indirect co-culture of RAW264.7 macrophages and 3T3-L1 adipocytes. 3T3-L1 preadipocytes were induced to mature in Transwell lower chamber, and then co-cultured with RAW264.7 macrophages in the upper chambers for 72 hours. Testosterone 10 μmol/L was added into indirect co-culture for 24 h. ELISA was used to testing IL-6, MCP-1 concentrations in supernatant. Western blot was used to detecting the phosphorylation of NF kappa B, ERK1/2, and theexpression of CD16/32 and CD206. Glucose transport was assessed by[H]2-deoxy glucose uptake in adipocytes. Testosterone enhanced inflammatory cytokines (IL-6, MCP-1) production in indirect co-culture of 3T3-L1 adipocytes and RAW264.7 macrophages, promoted the activation of ERK1/2 and nuclear factor kappa B p65, and inhibited glucose uptake in adipocytes. Testosterone facilitated the production of pro-inflammatory M macrophages. The above effects of testosterone can be completely reversed by PDTC, and can be partly reversed by PD98059 (70%-90%). NF kappa B and ERK1/2 could be the key proteins for testosterone to promote the production of inflammatory factors, to lead to insulin resistance, and to make macrophages differentiate to pro-inflammatory phenotypes in co-culture of RAW264.7 macrophages and 3T3-L1 adipocytes.