The active form of vitamin D (VD) exerts hormonal effects by regulating the expression of genes involved in T-cell activity, cell differentiation, and proliferation. Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of life-threatening diseases, adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy (HAM). Among ATL patients, hypercalcemia is one of the most serious complications due to bone resorption. In this study, wild-type mice administered UV-irradiated HTLV-1-infected cells showed up to 47% decrease of plasma VD level compared with untreated mice. To clarify the effect of HTLV-1 on plasma VD level, 315 samples registered in nationwide cohort study on ATL onset were measured. The VD level in HAM (14.98 ± 8.5 ng/mL) was significantly lower than those in asymptomatic carriers and ATL (p < 0.05). Upon comparing the VD levels in ATL stratified by disease subtypes, acute ATL showed a lower level (15.81 ± 12.0 ng/mL) than chronic and smoldering types (p < 0.05). In the longitudinal observation, VD levels were significantly higher in untreated spontaneous remission cases than in ATL progression cases, in which the VD levels decreased approximately 40% after onset. In cases of relapse after transplantation, the plasma VD level dropped to 38.7% of the pre-relapse level, while in cases of complete remission, the VD level increased with improvement of the performance status. Taken together, these results suggest that plasma VD level is a potential indicator for the onset and relapse of HTLV-1-associated diseases.