Type 2 diabetes mellitus (T2DM) is accompanied by insulin resistance in liver and peripheral tissue. Bacillus amyloliquefaciens fmb50 produces lipopeptide surfactin, which has a wide range of biological activities. However, the effects of surfactin on insulin resistance in any mouse model have not been reported. Our previous research demonstrated that dietary supplementation with surfactin can alleviate high fat diet (HFD)/ streptozotocin (STZ)-induced T2DM in mice by regulating intestinal microbiota. This study aims to explore the detailed mechanism of the effect of surfactin on liver and peripheral tissue. The results indicated that surfactin inhibited hepatic gluconeogenesis and promoted hepatic glycogen synthesis by activating the adenosine monophosphate-activated protein kinase and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) signalling pathways. Similarly, surfactin also significantly increased muscle glycogen synthesis by activating the PI3K/Akt signalling pathway. The glucose transporter 4 levels of skeletal muscle and adipose tissue, which are insulin-mediated vital organs, was noticeably increased after surfactin treatment. In addition, our results indicate that decreased hepatic fat accumulation and suppressed inflammation and oxidative stress in liver, skeletal muscle and adipose tissue also play critical roles in ameliorating insulin resistance. These findings demonstrate that surfactin not only ameliorates liver dysfunction but also enhances insulin sensitivity in HFD/STZ-induced T2DM mice. Finally, using fasting blood glucose, oral glucose tolerance test and insulin tolerance test, we show that early surfactin intervention can mitigate T2DM in mice. The study results warrant future functional food studies which will provide insight into diet-host interactions.