Vacuolization of the cytoplasm is one of the dramatic and frequently observed phenomena in various cell types. Cellular vacuoles occur spontaneously or via a wide range of inductive stimuli, but the molecular mechanism involved in this process remains largely unknown. In this study, we investigated the role of the p38 and JNK pathways in the formation of cytoplasmic vacuoles. We found that p38 and JNK agonist anisomycin abolishes spontaneous cytoplasmic vacuolization of HepG2 cells through p38 activation, but not through JNK activation. Importantly, blocking the activity of p38 or suppression the expression of p38 elicits cytoplasmic vacuoles formation in various cancer cells. Furthermore, cytoplasmic vacuoles induced by p38 blocking are derived from the perinuclear region. These observations provide direct evidence for a role of p38 signaling in regulating the formation of cytoplasmic vacuoles. J. Cell. Biochem. 114: 1789–1799, 2013. © 2013 Wiley Periodicals, Inc.