Previously, we reported that repeated oral administration of etoposide (ETP) increases P-glycoprotein (P-gp) expression in association with activation of ezrin/radixin/moesin (ERM) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) signaling in the small intestine. However, the detailed mechanisms of this pathway have yet to be fully elucidated. Recently, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], one of the most abundant phosphoinositides in the plasma membrane, has attracted attention regarding its involvement in the plasma membrane localization of various membrane proteins. PtdIns(4,5)P2 is an essential factor in the dissociation and subsequent membrane translocation (activation) of ERM, and its synthetic pathway is known to be highly regulated by RhoA/ROCK signaling. Here, we examined the involvement of PtdIns(4,5)P2 in the mechanism by which ETP treatment increases small intestinal P-gp levels, and we determined which protein within ERM contributes to this phenomenon. Repeated oral treatment with ETP (10 mg/kg/d) over 5 d significantly increased PtdIns(4,5)P2 expression in the ileal membrane as measured by dot blot. Furthermore, this increase was suppressed by co-administration of a RhoA inhibitor, rosuvastatin (5 mg/kg/d, per os (p.o.)), or a ROCK inhibitor, fasudil (5 mg/kg/d, p.o.). In immunoprecipitation assays, radixin (but not ezrin or moesin) binding to PtdIns(4,5)P2 was observed to increase in association with the up-regulation of P-gp in the same fraction, and immunofluorescence studies indicated that radixin co-localized with PtdIns(4,5)P2 in the ileal tissue. In conclusion, ETP treatment appears to up-regulate PtdIns(4,5)P2 expression via RhoA/ROCK signaling, leading to the activation of ERM, presumably through the physical interaction of radixin with PtdIns(4,5)P2. This in turn increases the expression of ileal P-gp.