Side effects of methotrexate (MTX) especially hepatotoxicity limits clinical applications of this anticancer agent. Carboxypeptidase G2 (CPG2) is administrated for the treatment of elevated plasma concentrations of MTX. In this study, we have investigated the intracellular delivery of CPG2 fused to the transactivator transduction domain (TAT) and its protective effects against MTX-induced cell death of HepG2 cells. We have observed that both native and denatured forms of the enzyme transduced into the HepG2 cells efficiently in a concentration and time-dependent manner. The denatured protein transduced with higher efficiency than the native form and was functional inside the cells. MTX exposure significantly decreased HepG2 cell viability in a dose- and time-dependent manner. The cell viability after 24 and 48h of incubation with 100μM MTX was reduced to 44.37% and 17.69%, respectively. In cells pretreated with native and denatured TAT-CPG2 protein the cell viability was 98.63% and 86.31% after 24 and 48h, respectively. Treatment with MTX increased the number of apoptotic HepG2 cells to 90.23% after 48h. However, the apoptosis percentage in cells pretreated with native and denatured TAT-CPG2 was 21.49% and 22.28%, respectively. Our results showed that TAT-CPG2 significantly prevents MTX-induced oxidative stress by decreasing the formation of ROS and increasing the content of glutathione (GSH) and catalase activity. Our finding indicates that both native and denatured TAT-CPG2 strongly protect HepG2 cells against MTX-induced oxidative stress and apoptosis. Hence, intracellular delivery of CPG2 might provide a new therapeutic strategy for protecting against MTX mediated cytotoxicity.