Coxsackievirus B3 (CVB3) is a common human pathogen associated with many diseases. The present paper aimed to investigate that the inhibitory effect of chaetocin, a metabolite isolated from Chaetomium, on CVB3 replication in vitro. Treatment of HeLa cells with 250 nmol/L chaetocin increased the relative cell viability from (21.9±1.8)% to (70.1±4.3)%, while the production of progeny virus in chaetocin-treated cells was only (5.3±0.8)% of that in untreated cells. The viral RNA level in chaetocin-treated cells was also reduced to about (13±8.3)% of the control. Chaetocin treatment also increased the relative cell viability of CVB3-infected Vero cells from (64.6±1.7)% to (87.2±4.8)%. This inhibitory effect of chaetocin was partially remediated by the antioxidant agent N-acetyl cysteine. Further analysis of the mechanism may help the development of antiviral drug against CVB3 and other enteroviruses.