Adult T-cell leukemia (ATL) is one of the most aggressive hematologic malignancies caused by human T-lymphotropic virus type 1 (HTLV-1) infection. The prognosis of ATL is extremely poor; however, effective strategies for diagnosis and treatment have not been established. To identify novel therapeutic targets and diagnostic markers for ATL, we employed focused proteomic profiling of the CD4(+)CD25(+)CCR4(+) T-cell subpopulation in which HTLV-1-infected cells were enriched. Comprehensive quantification of 14 064 peptides and subsequent 2-step statistical analysis using 29 cases (6 uninfected controls, 5 asymptomatic carriers, 9 HTLV-1-associated myelopathy/tropical spastic paraparesis patients, 9 ATL patients) identified 91 peptide determinants that statistically classified 4 clinical groups with an accuracy rate of 92.2% by cross-validation test. Among the identified 17 classifier proteins, α-II spectrin was drastically accumulated in infected T cells derived from ATL patients, whereas its digestive protease calpain-2 (CAN2) was significantly downregulated. Further cell cycle analysis and cell growth assay revealed that rescue of CAN2 activity by overexpressing constitutively active CAN2 (Δ(19)CAN2) could induce remarkable cell death on ATL cells accompanied by reduction of α-II spectrin. These results support that proteomic profiling of HTLV-1-infected T cells could provide potential diagnostic biomarkers and an attractive resource of therapeutic targets for ATL.