Background: Colorectal cancer (CRC) represents an important neoplasm with high mortality. Although PD-L1/PD-1 system-based immunotherapy has benefits for a certain type of CRC, many efforts should be made to enhance the responses to anti-PD-1/PD-L1 drugs. DNA methylation has been critically implicated in the regulation of tumor immunity. Here, we examined the effects of the natural alkaloid oxymatrine on PD-L1 expression in CRC cells and to elucidate the underlying mechanism. Methods: Human CRC SW620 and HCT116 cells were treated with interferon γ (IFNγ) and/or oxymatrine. Cell viability was determined using MTT assays. PD-L1 expression was detected by real-time PCR and Western blot analyses. DNA demethylase activity was measured using kits. Results: Oxymatrine did not apparently affect the viability of normal human intestinal epithelial cells. IFNγ at 20 ng/ml increased the viability of CRC cells, but oxymatrine concentration-dependently reduced the viability in the absence or presence of IFNγ. IFNγ increased the mRNA and protein expression of PD-L1 in the two cell lines, but oxymatrine significantly abolished IFNγ-elevated PD-L1 levels at both mRNA and protein levels. Furthermore, DNA demethylase activity was remarkably increased in IFNγ-treated CRC cells, which was abolished by oxymatrine concentration-dependently. In addition, DNA methyltransferase inhibitor 5-azacytidine considerably abrogated oxymatrine-induced downregulation of PD-L1 mRNA and protein levels in IFNγ-stimulated CRC cells. Conclusion: Oxymatrine suppressed viability and reduced PD-L1 expression in IFNγ-stimulated CRC cells, which was attributed to enhanced DNA demethylation. Our current discoveries suggested oxymatrine as an epigenetic modulatory agent for immunotherapy against CRC via PD-1/PD-L1 blockade. © 2020, Federación de Sociedades Españolas de Oncología (FESEO).