Retrochalcone compound, a licochalcone C (LCC) originating from the root of Glycyrrhiza inflata, has shown anticancer activity against esophageal squamous cell carcinoma, skin cancer, and oral squamous cell carcinoma. However, the therapeutic potential of LCC in treating colorectal cancer (CRC) and its underlying molecular mechanisms remain unclear. In this study, we examined the anti-proliferative activity of LCC in human colorectal cancer cells (HCT116), oxaliplatin (Ox) sensitive and Ox-resistant colorectal cancer cells (HCT116-OxR). LCC significantly and selectively inhibited the growth of HCT116 and HCT116-OxR cells. An in vitro kinase assay showed that LCC inhibited the kinase activities of EGFR and AKT. Decreased phosphorylation of EGFR and AKT was observed in the LCC-treated cells. In addition, LCC induced cell cycle arrest by modulating the expression of cell cycle regulators cyclin B1 and cdc2. Moreover, LCC treatment dysregulated mitochondrial membrane potential (MMP), and the disruption of MMP resulted in the release of cytochrome c into the cytoplasm and activation of caspases to execute apoptosis. Overall, LCC showed anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting EGFR and AKT, inducing ROS generation and disrupting MMP. Thus, LCC may be potential therapeutic agents for the treatment of Ox-resistant CRC cells.