Pan-Bcl-2 family inhibitor obatoclax has been demonstrated to be effective against various cancers, of which the mechanism of action is not fully understood. In this study, we demonstrate that obatoclax suppressed esophageal cancer cell viability with concomitant G1/G0-phase cell cycle arrest. At the tested concentrations (1/2 IC50 and IC50), obatoclax neither induced PARP cleavage nor increased the Annexin V-positive population, suggesting G1/G0-phase arrest rather than apoptosis accounts for most of the reduction of cell viability produced by obatoclax. Double knockdown of Bak and Bax by small interference RNA failed to block obatoclax-induced G1/G0-phase arrest, implying its role in cell cycle progression is Bak/Bax-independent. The cell cycle arresting effect of obatoclax was associated with up-regulation of p21waf1/Cip1. Knockdown of p21waf1/Cip1 significantly attenuated obatoclax-induced G1/G0-phase arrest. Although obatoclax stimulated phosphorylation of Erk, p38, and JNK, pharmacological inhibition of p38 but not Erk or JNK blocked obatoclax-induced G1/G0-phase arrest. Moreover, knockdown of p38 abolished the cell cycle arresting effect of obatoclax. In consistent with this finding, inhibition of p38 blocked obatoclax-induced p21waf1/Cip1 expression while inhibition of Erk or JNK failed to exert similar effect. To conclude, these findings suggest that obatoclax induced cell cycle arrest via p38/p21waf1/Cip1 signaling pathway. This study may shed a new light on the anti-cancer activity of obatoclax in relation to cell cycle arrest. J. Cell. Biochem. 115: 1624–1635, 2014. © 2014 Wiley Periodicals, Inc.