Obese adipose tissue is characterized by adipocyte hypertrophy and a massive macrophage infiltration. The interaction between macrophages with mature adipocytes releases pro‐inflammatory cytokines. This chronic inflammatory state can contribute to obesity‐related complications, such as insulin resistance, type 2 diabetes and cardiovascular disease. Therefore, we can attempt to prevent and treat obesity‐related diseases by inhibiting macrophage infiltration and blocking their interaction with adipocytes. Honey bee (Apis mellifera) venom (BV) has been reported to have anti‐inflammatory effects. Although BV is used to treat chronic inflammatory diseases, few studies have addressed its use in obesity‐associated inflammation. This study examines the inhibitory effects of BV on lipid accumulation in differentiating preadipocytes, inflammation, and insulin resistance in macrophages and adipocyte‐macrophage co‐culture system. We treated 3 T3‐L1 preadipocytes with BV during differentiation. We later measured lipid accumulation and gene expression of master adipogenic transcription factors. After RAW264.7 and 3 T3‐L1 cells were pretreated with BV, RAW264.7 cells were activated with LPS or co‐cultured with pretreated 3 T3‐L1 cells. Gene expression of pro‐inflammatory cytokines and insulin sensitizing genes was measured in these cells. BV inhibited lipid accumulation and C/EBPα and PPARγ gene expression during intermediate and late 3 T3‐L1 cell differentiation. BV also suppressed gene expression of pro‐inflammatory cytokines (COX‐2, iNOS, MCP‐1, TNF‐α, IL‐1β and IL‐6) in LPS‐stimulated macrophages, and in co‐culture of 3 T3‐L1 adipocytes and RAW264.7 macrophages. However, adiponectin and GLUT‐4 expression were both significantly increased by BV in co‐culture. These findings demonstrate that BV attenuates adipocyte hypertrophy and improves obesity‐related inflammation and insulin resistance in obese adipose tissue.