Introduction: Cyclosporine A (CsA), a calcineurin inhibitor, causes chronic nephrotoxicity during organ transplantation. Objective: The aim is to investigate the effect of ethyl acetate fraction (EF) of Boerhavia diffusa on CsA-induced cell damage due to apoptosis and reactive oxygen species (ROS). Materials and Methods: Madin–Darby Canine Kidney (MDCK) cells were treated with CsA alone and CsA plus EF (25 and 50 μg/ml). The mechanism of attenuation of apoptosis and ROS by EF was studied using various experiments. Results: EF protected the MDCK cells from CsA-induced apoptosis and cell cycle arrest at G0/G1and sub G0/G1. The protective effect was further confirmed by cytochrome c translocation into cytoplasm and caspase 3 release. In addition, CsA-induced ROS production was abolished by the antioxidant potential of EF, which could be attributed to the polyphenol content. The decrease in ROS generation was confirmed by DCF-DA staining, LPO, and nitric oxide release in the MDCK cells. Moreover, the accumulation of collagen was decreased in EF-treated groups and increased the survivability of cells. The gas chromatography–mass spectrometry analysis revealed the presence of alkaloid and phenolic compounds. To the best of our knowledge, this is the first report showing the presence of alkaloid Carnegine and N-Benzyl-2-phenethylamine in this plant. Conclusion: Hence, it could be justified that EF-protected MDCK cells against CsA stimulated renal cell damage by attenuating apoptosis and ROS generation. Further isolation of compound has to be carried out to explore the effectiveness of EF as nephroprotective drug.