Aim To construct the lentivirus expression vector of overexpression for RagB gene, realize the overexpression of RagB gene in C2C12 cells, test the regulation of mammalian target of rapamycin (mTOR) activation mediated by Ursolic acid. Methods The lentivirus expression vectors, Flag pLJM1 RagBWT, Flag pLJM1 RagB⁵⁴L and Flag pLJM1 RagB⁹⁹L were employed in the experiment. Using PCR to amplify the full length of Flag-RagBWT, Flag-RagB⁵⁴L and Flag-RagB⁹⁹L, then they were subcloned into the pWPI lentivirus vector through SwaI restriction site and identified by bacteria liquid PCR. The cloned lentivirus vectors(pWPI-RagBWT, pWPI-RagB⁵⁴L and pWPI-RagB⁹⁹L) were packaged with packaging vectors (psPAX2 and pMD2.G) via specific ratio and transfected into 293T cells. 48 hours after the transfection, the media was collected and used to infect C2C12 cells. RagB⁹⁹L-overexpressed C2C12 myoblasts were treated individually or jointly with leucine and ursolic acid, mTOR activation was detected by Western blot. Results The efficiency of infection observed by fluorescence microscope was almost 100%, the expression of RagB in C2C12 cells proved by Western blot was enhanced significantly in RagB overexpression group. Ursolic acid inhibited significantly mTOR activation in RagB⁹⁹L-overexpressed C2C12 myoblasts. Conclusion The results indicate that RagB can be overexpressed in C2C12 cells by constructing the lentivirus expression vector, RagB plays a critical role in ursolic acid-inhibited mTOR activation.