Arsenic trioxide (As(2)O(3)) has shown substantial efficacy in the treatment of patients with acute promyelocytic leukemia, a specific subtype of acute myeloid leukemia (AML). However, since not all patients can achieve remission after treatment, it is necessary to develop a novel method to overcome this problem. We investigated the anti-leukemic effect of low-dose 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) in combination with As(2)O(3) on the human AML cell lines HL-60 and K562. The cell viability was in reverse proportion to As(2)O(3) or 1,25(OH)(2)D(3) concentration. In both HL-60 and K562 cells, after the combination treatment with As(2)O(3) and 1,25(OH)(2)D(3) at a 10:1 ratio, the combination index (CI) values were <1 in all treatment groups. In the RT-PCR and western blot analysis, the combination treatment decreased Bcl-2 expression and increased Bax and caspase-3 expression more prominently than the single treatment. In the flow cytometric analysis performed in HL-60 cells, the proportion of late apoptotic cells was 4.9% in the control, 30.0% in cells treated with 1.0 µM As(2)O(3), 8.1% in cells treated with 100 nM 1,25(OH)(2)D(3), and 64.3% in cells treated with 1.0 µM As(2)O(3) plus 100 nM 1,25(OH)(2)D(3). In conclusion, low-dose 1,25(OH)(2)D(3) combined with As(2)O(3) synergistically inhibited proliferation of HL-60 and K562 cells. In addition, this combination activated the apoptosis pathway more prominently than the single-drug treatment.