Protein phosphatase 2A (PP2A) is a widely conserved protein serine/threonine phosphatase that is implicated in regulating a wide array of cellular functions including apoptosis. The present study tests the hypothesis that induced TRPC1 expression inhibits NF-B activation by increasing PP2A activity through Ca²⁺ influx in IECs. The expression of TRPC1 induced by stable transfection with the wild-type TRPC1 gene increased PP2A activity as indicated by increases in levels of PP2A proteins and their phosphatase activity. Increased levels of PP2A activity in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1) were associated with decreased nuclear levels of NF-B proteins and a reduction in NF-B dependent transcriptional activity, although there were no changes in total NF-B protein levels. Inhibition of PP2A activity by treatment with okadaic acid or PP2A silencing with small interfering RNA not only enhanced NF-B transactivation but also prevented the increased susceptibility of IEC-TRPC1 cells to apoptosis induced by treatment with tumor necrosis factor- (TNF-)/cycloheximide (CHX). Decreasing Ca²⁺ influx by exposure to the Ca²⁺-free medium reduced PP2A mRNA levels, destabilized PP2A proteins, and induced NF-B activation, thus blocking the increased sensitivity of IEC-TRPC1 cells to TNF-/CHX-induced apoptosis. These results indicate that induced TRPC1 expression increases PP2A activity through Ca²⁺ influx and that increased PP2A sensitizes IECs to apoptosis as a result of NF-B inactivation.