We examined the effects of transforming growth factor β₁ (TGFβ₁) on cellular functions in human lung cancer cell line A549. Treatment of A549 cells with 1 ng/ml TGFβ₁ for more than 3 days altered their morphology from an epithelial cobblestone-like appearance to a fibroblast-like one, reduced the expression of E-cadherin mRNA and protein, and induced the formation of F-actin fibers. These hallmarks indicate that TGFβ₁ induced the epithelial–mesenchymal transition in A549 cells. Migration of TGFβ₁-treated A549 cells, which was quantified by the wound-healing assay, was markedly accelerated by 3 μM ATPγS, a non-hydrolyzable ATP analogue. ATPγS-induced migration of TGFβ₁-treated A549 cells was reversed by the P2 antagonist suramin. In contrast, migration of control A549 cells was not altered by ATPγS. TGFβ₁-treated A549 cells showed an augmentation of ATP-induced Ca²⁺ transients, thapsigargin-induced Ca²⁺ transients, and store-operated Ca²⁺ entry compared with those in control cells. Basal level of the extracellular ATP concentration was significantly lower in TGFβ₁-treated A549 cells than in control cells. We conclude from these results that TGFβ₁ augments ATP-induced Ca²⁺ mobilization, which leads to the acceleration of migration, in A549 cells but, it markedly reduces endogenous ATP release. This implies that the actions of ATP would become a novel therapeutic target for inhibiting cancer cell migration.