〔Abstract〕 Objective To investigate the effect of palmitoleic acid(POA) on pyroptosis of cardiomyocytes after hypoxia/reoxygenation-induced injury in the human myocardium. Methods The experiment comprised a control group(Control, normal culture), a hypoxia/reoxygenation group(HR), a palmitoleic acid-treated group(HR+POA), and an anhydrous ethanol control group(HR+ET). Cardiomyocytes viability was assessed using CCK-8 assay, and the level of pyroptosis of cardiomyocytes was measured through the double staining with Hoechst33342/PI and LDH assay. ELISA was employed to detect the release of inflammatory factors IL-1β and IL-18 in the cell culture supernatant. qRT-PCR and Western blot were utilized to determine the relative expression levels of mRNA and protein of pyroptosis-related genes, namely NLRP3, ASC, Caspase-1, GSDMD, IL-1β and IL-18, respectively. Results CCK-8 assay showed that the survival of hypoxic/reoxygenated cardiomyocytes increased with the addition of POA at concentrations ranging from 25 to 100 μmol/L, as compared to the HR group; a hypoxia/reoxygenation model of cardiomyocyte was established. The expression of protein and mRNA increased in NLRP3, ASC, Cleaved caspase-1, GSDMD-N, IL-Iβ and IL-18vsthe control group(P<0.05), the positive percentage of Hoechst33342/PI staining in cardiomyocytes increased significantly(P<0.05), the release of LDH, IL-Iβ, and IL-18 increased(P<0.05). After intervention with 100 μmol/L POA, the protein and mRNA expression levels of NLRP3, ASC, Cleaved caspase-1, GSDMD-N, IL-Iβ, and IL-18 were significantly reduced in the HR+POA group vs HR+ET group(P<0.05).The positive percentage of Hoechst33342/PI staining in cardiomyocytes decreased significantly, and the levels of LDH, IL-Iβ and IL-18 significantly decreased(P<0.05). Conclusion Palmitoleic acid may alleviate hypoxia/reoxygenation-induced injury of cardiomyocytes by inhibiting pyroptosis and inflammatory response after hypoxia/reoxygenation in human myocardium.