Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in 2-dimensional (2D) and 3-dimensional (3D) culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on Geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both Farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced more cell death in RH30 cells compared to C2C12 in 3D culture model with similar effects on autophagy flux as in 2D culture. Overall our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin myotoxicity is different in 2D versus 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.