Objective To investigate the protective effect of morroniside (MR) on myocardial injury induced by Coxsackievirus B3 (CVB3) and its mechanism. Methods Cardiomyocytes from SD mice were isolated and cultured, and myocardial inflammatory injury model was established by infecting cardiomyocytes with coxsackievirus B3 (CVB3). Cardiomyocytes were randomly divided into 5 groups: Control group, CVB3 group, and CVB3+MR (10, 50, and 100 μM) group. CCK-8 was used to detect cardiomyocyte viability, and Western blot was used to detect the expression of NLRP3, ASC, Cleaved caspase-1, Cleaved caspase-3, Caspase-9, Bcl-2, and Bax proteins. ELISA was used to detect IL-1β, IL-18, CK-MB IL-1β, IL-18, CK-MB and cTnI were detected by ELISA. Results There was no difference in the effect of MR's on cardiomyocyte viability in each dose group (P＞0.05). The expression of cardiac damage markers CK-MB and cTnI as well as inflammatory factors IL-1β and IL-18 was increased in the CVB3 group compared with the Control group (P＜0.05), and all of the above indicators were decreased in the CVB3+MR group compared with the CVB3 group (P＜0.05). Compared with the Control group, the expression of NLRP3, ASC, Cleaved caspase-1, Cleaved caspase-3, caspase-9 and Bax was increased in the CVB3 group (P＜0.05), and the expression of Bcl-2 was decreased in the CVB3+MR group (P＜0.05), and compared with the CVB3 group, the expression of Bcl-2 was increased (P＜0.05), and all other protein expressions decreased (P＜0.05).Conclusion MR attenuates inflammatory injury in CVB3-infected cardiomyocytes by mechanisms related to the inhibition of myocardial charring, apoptosis, and inflammatory responses