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The failure of and resistance to oxaliplatin (OXA)-based chemotherapies may lead to poor prognosis in colorectal cancer (CRC) patients. It has been reported that orlistat (Orli) exhibits potent antitumor effects in several malignant tumors. Here, we identified that OXA in combination with low-dose Orli could sensitize CRC cells to OXA and induce marked synergistic apoptosis in vitro and in vivo. The potential synergistic effects were confirmed and quantified by in silico analysis. Furthermore, we validated the synergistic anti-tumor effects in CRC PDX mice model. A qPCR array was performed to evaluate the changes in 85 apoptosis-related genes to elucidate the possible molecular mechanisms in combination-induced cytotoxicity. To conclude, the antitumor synergistic effects of OXA and Orli make them effective and promising candidates for cancer treatment.