Abstract: Objective: To investigate whether lactate/GPR81 signal pathway is involved in the regulation of skeletal muscle contraction-induced lipid droplets accumulation. Methods: C2C12 cells were treated with lactate firstly, then the cells' triglyceride (TG) content was determined by colorimetric method. The expression levels of CREB, pCREB and β-actin protein were detected by Western Blot. After that, C2C12 cells were treated by two kinds of electric pulse stimulation (EPS) protocol (i.e., 30 V 0.5 Hz 2 ms and 30 V 1 Hz 2 ms) . The content of lactate was determined by colorimetric method, the level of ATP was detected by chemiluminescence method, and the mR NA level of MyHCI, MyHCIIa, MyHCIIb and MyHCIIx were detected by RT-PCR. Subsequently, the changes of TG content in C2C12 myotubes at different time points after EPS treatment were observed, in order to establish the model of EPS-induced TG accumulation in C2C12 myotubes. Finally, the cells were treated with GPR81 inhibitor 3-hydroxy-butyrate (3-OBA) and divided into six groups: control group, 30 V 0.5 Hz 2 ms group, 30 V 1 Hz 2 ms group, 3-OBA group, 3-OBA+30 V 0.5 Hz 2 ms group and 3-OBA+30 V 1 Hz 2 ms group. The content of TG was detected by chemiluminescence method. Results: 1) Compared with control group, the lactate increased the content of TG in C2 C12 myotubes (P<0.05) and decreased the pCREB signaling status (P<0.01) significantly; 2) Compared with control group, two EPS protocols increased the lactate concentration (P<0.05) and reduced the ATP content (P<0.01) in C2C12 cells significantly; the mRNA levels of MyHCI and MyHCIIb were significantly increased in these two groups, but the mRNA level of MyHCIIa and MyHCIIx were unchanged (P>0.05) ; the TG content was significantly increased at 36 h after EPS (P<0.05) , but the TG content was unchanged at 12 h and 24 h (P>0.05) ; 3) Compared with non-EPS group, the TG content in 30 V 0.5 Hz 2 ms group was increased (P=0.052) , but there was no significant change when the 3-OBA was applied (P>0.05) ; the TG content in 30 V 1 Hz 2 ms group was significantly increased (P<0.05) , but the TG content was significantly reduced after the treatment of 3-OBA (P<0.05) . Conclusions: Long-time EPS induced the contraction of the myotubes and increased the contents of lactate and TG. Lactate/GPR81 signaling pathway mediated the C2C12 myotubes cell contraction-induced TG accumulation, and it is one of the molecular mechanisms in skeletal muscle cell contraction-induced lipid droplets accumulation of energy substrate.