Surfactin, produced by fmb50, was used to treat insulin-resistant (IR) hepatocyte. It was found that surfactin increased glucose consumption in insulin-resistant HepG2 (IR-HepG2) cells and ameliorated IR by increasing glucose transporter 4 (GLUT4) protein expression and AMP-activated protein kinase (AMPK) mRNA expression, promoting GLUT4 translocation and activating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) in IR-HepG2 cells. Meanwhile, surfactin downregulated protein expression of phosphoenolpyruvate carboxy kinase (PEPCK) and glucose-6-phosphatase (G6Pase), further inhibiting hepatic gluconeogenesis. In addition, surfactin played important roles in eliminating reactive oxygen species (ROS), improving mitochondrial dysfunction, and inhibiting proinflammatory mediators. We observed that surfactin promoted glucose consumption, meanwhile increased translocation and protein expression of GLUT4 in Caco-2 cells. These results confirmed the conclusion in hepatic cells. Furthermore, surfactin supplement decreased body weight, food intake, and fasting blood glucose of type 2 diabetes mellitus (T2DM) mice induced by streptozotocin (STZ)/high-fat diet (HFD). Our data indicated that surfactin ameliorated insulin resistance and lowered blood glucose in intro and in vivo.