Lymphoma is one of the most common malignant tumors in canine. Protein phosphatase 2A (PP2A), a well-conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. Perphenazine (PPZ) is one of the phenothiazines and widely used as an antipsychotic drug. Recently, it is reported that PPZ directly binds with scaffolding subunit of PP2A complex and exerts anti-tumor effects on human T cell acute lymphoblastic leukemia. However, the effect of PPZ on canine lymphoma has not been studied. Here, we investigated the potential therapeutic role of PPZ and its molecular mechanism in canine T-cell lymphoma. In canine T-cell lymphoma cell lines, UL-1 and Ema, PPZ decreased cell survival in a dose-dependent manner. Increased caspase 3 activity and Annexin V positive cells suggested that PPZ induced apoptosis. PPZ dephosphorylated Akt, MEK1/2 and ERK1/2. Akt inhibitor, but not MEK1/2 inhibitor and ERK1/2 inhibitor, induced cell death, indicating the importance of Akt dephosphorylation for the anti-tumor effect of PPZ. Finally, we observed enhanced PP2A activity by PPZ treatment. The present results for the first time revealed that PPZ induced canine lymphoma cells apoptosis through Akt dephosphorylation via PP2A activation. Our study suggests the possible therapeutic application of phenothiazines for canine T-cell lymphoma.