Transforming growth factor-β (TGF-β) suppresses growth via the TGF-β-SMAD pathway but promotes growth in cancer cells with disrupted SMAD signaling and corresponds to an invasive phenotype. TGF-β also downregulates the tumor suppressor PTEN that is rarely mutated in sporadic pancreatic cancer; this downregulation may mediate cell proliferation and invasiveness, but the mechanism is unknown. Here, we examined whether TGF-β modulation of PTEN was mediated by protein kinase C (PKC). We have previously demonstrated that SMAD4-null BxPc-3 pancreatic cancer cells treated with TGF-β1 (10 ng/ml) suppressed PTEN expression and increased cell proliferation. TGF-β-treated cells were examined for PKC activation and its coupling to PTEN expression, utilizing pharmacological and knockdown methods. Calcium mobilization and cell migration were also examined. In BxPc-3 cells, only two PKC isoforms were activated by TGF-β, and PTEN downregulation by TGF-β was specifically mediated by PKC-α. In parallel, TGF-β rapidly induced an increase in cytoplasmic free calcium from intracellular stores, consistent with subsequent PKC-α activation. The TGF-β-induced increase in cell migration was blocked by knockdown of PKC-α. Thus calcium-dependent PKC-α mediates TGF-β-induced transcriptional downregulation of PTEN, and this pathway promotes cell migration in a SMAD4-null environment. The TGF-β-PKC-α-PTEN cascade may be a key pathway for pancreatic cancer cells to proliferate and metastasize.