Osteoporosis is characterized by the systemic impairment of bone mass, strength, and microarchitecture, leading to an increased risk of fragility fracture. Bisphosphonates (BPs) are the first-line drugs for osteoporosis. Vitamin D is considered to be essential for osteoporotic treatment. However, long-term effects of BPs on the serum levels of 25-hydroxyvitamin D 3 (25(OH)D 3 ) are unknown. Accordingly, in this retrospective study, we collected clinical data of 41 post-menopausal Japanese women with osteoporosis treated with BP for over 3 years, without vitamin D supplementation. We measured lumbar and femoral neck bone mineral density (BMD) and serum levels of bone specific alkaline phosphatase (BAP) as a bone formation marker, and tartrate-resistant acid phosphatase (TRACP)-5b as a bone resorption marker, before and after the 3-year treatment. Serum 25(OH)D 3 , 1,25(OH) 2 D 3 , and whole parathyroid hormone (PTH) were also measured. Notably, no fracture occurred during the treatment. Compared with baseline values, 25(OH)D 3 levels were significantly increased from 21.6 to 26.4 ng/mL (P = 0.006), despite no vitamin D supplementation. 1,25(OH) 2 D 3 and whole PTH levels tended to be decreased from 62.6 to 57.8 pg/mL and 27.3 to 25.1 pg/mL, respectively. Both bone formation and resorption markers were significantly suppressed (P < 0.01). Both lumbar BMD (7.3% increase) and femoral neck BMD (4.1% increase) were significantly improved (P < 0.0001) after 3 years of the treatment. Thus, even without vitamin D supplementation, serum 25(OH)D 3 levels were significantly increased after 3-year BP therapy. These results suggest that vitamin D supplementation might not be required in the long-term BP therapy for osteoporosis.