Vitamin D insufficiency and deficiency are common in the elderly. Most previous studies using alendronate have used vitamin D supplementation regardless of individual vitamin D status. However, the minimum required vitamin D levels for the efficacy of alendronate treatment of osteoporosis remain unclear. Fifty-two postmenopausal women, diagnosed with osteoporosis, were enrolled in this prospective study, in which they took 5 mg of alendronate daily for 6 months without any supplements. Associations between baseline factors and their changes during the treatment and the change in the lumbar spine bone mineral density (LS-BMD) were examined. The most appropriate cut-off level of 25-hydroxyvitamin D (25[OH]D) for the optimal increase in LS-BMD with alendronate was determined using the Akaike information criterion statistical criterion. Overall, alendronate treatment significantly increased LS-BMD by 4.7%. The basal serum 25(OH)D and change in urinary NTX were significantly associated with the increase in LS-BMD. The increase in LS-BMD between the two groups was not different when comparing those with baseline 25(OH)D above vs. below 30 ng/ml. However, 25(OH)D of 25 ng/ml was determined to be the minimum required vitamin D level for an adequate effect of alendronate. Vitamin D status may affect the increase in LS-BMD with alendronate treatment in individuals being treated for osteoporosis, and a 25(OH)D level >25 ng/ml appears to be required for an optimal LS-BMD response.