Pre-transplant insulin resistance has been proved to be an important risk factor for organ transplantation, predicting increased post-transplant complications and worse survival outcomes. However, the underlying mechanism is still unclear. Cyclosporin A (CsA) is widely used as an immunosuppressant after organ transplantation, while emerging evidence has shown that CsA increases the risk of post-transplant complications. Thus, in this study, using a cellular model of palmitate-induced insulin resistance, we evaluate the effect of CsA on apoptosis in skeletal muscle C2C12 cells with palmitate-induced insulin resistance. Western blot and flow cytometric analysis showed that CsA induced apoptosis in insulin-resistant C2C12 cells. Mechanistically, a sustained increase of global protein O-GlcNAcylation was observed after CsA treatment, and suppression of protein O-GlcNAcylation with its inhibitors (alloxan or 5-oxo-6-diazo-norleucine) resulted in decreased O-GlcNAcylation levels and apoptosis. Furthermore, CsA increased mitochondrial membrane potential and intracellular ROS production in insulin-resistant C2C12 cells, and inhibition of ROS production with SS-31 suppressed CsA-induced O-GlcNAcylation. In summary, our results suggest that CsA treatment induced apoptosis in insulin-resistant C2C12 cells, partly via CsA-induced ROS production and resultant O-GlcNAcylation, indicating that O-GlcNAcylation serves as a potent therapeutical target for organ transplantation.