Retinol-binding protein 4 (RBP4) is adipocyte-derived secreted adipokines and elevated RBP4 expression level was closely related to insulin resistance and type II diabetes mellitus. However, the exact mechanisms are unknown. To clarify the mechanism, RBP4 lentivirus particles were packaged to infect porcine preadipocytes. Then procine preadipocytes were activated by insulin or induced model of insulin resistance. RBP4 interference efficiency and the gene expression of each treatment groups in PI3K/Akt pathways were examined by QRT-PCR and Western blotting. The result shows that RBP4 mRNA and protein expressions were suppressed more than 60% (P<0.01). Furthermore, no matter under insulin stimulation or insulin resistance, RBP4 knockdown significantly increased the mRNA expressions of AKT2, PI3K, GLUT4 and IRS1 compared with the control. The protein phosphorylate levels of AKT2, PI3K, IRS1 arised, meanwhile enhanced the AKT2, PI3K, GLUT4 total protein expressions. Collectively, knockdown of RBP4 increased the insulin sensitivity through upregulated PI3K/Akt pathways related factors’ expression and phosphorylation in porcine adipocytes. This research will provide a new idea to treat insulin resistance related diseases.