Photobiomodulation (PBM) could improve systemic blood glucose and insulin resistance in diet-induced diabetic mice. A few possible molecular mechanisms for the beneficial effects of PBM on diabetes have been proposed, but there is still an urgent need to explore the underlying mechanisms that support the application of PBM in the treatment of diabetes. Our study aimed to evaluate the effects of PBM on lipid metabolism in the liver of high-fat diet (HFD)-induced mice and explore the potential mechanisms of PBM on obesity and type 2 diabetes. Here, we administered PBM therapy (wavelength: 635 nm, energy density: 8 J/cm) daily for eight weeks to HFD-induced mice. We detected that eight-week daily administration of PBM ameliorated HFD-induced gain weight, hyperlipidemia, and hyperglycemia, but also protected against diet-induced hepatic steatosis and insulin resistance. Furthermore, PBM increased AMP-activated protein kinase (AMPK) activation, lowered nuclear translocation of sterol regulatory element binding protein 1 (SREBP1), decreased aberrant lipogenesis, and enhanced insulin sensitive in HFD-induced mice livers. We also observed that Ca/calmodulin-dependent protein kinase kinase β (CaMKKβ) activation was responsible for AMPK activation in insulin-resistant HepG2 cells exposed to PBM. In summary, PBM at 635 nm and 8 J/cm improved hepatic lipid metabolism and inhibited the development of HFD-induced obesity and type 2 diabetes. Moreover, increased intracellular Ca content and CaMKKβ-dependent AMPK activation were possible molecular mechanisms underlying the PBM-induced improvement on obesity and type 2 diabetes.Copyright © 2020. Published by Elsevier B.V.