The aim of this study was to evaluate the effects of a combination of serum 25-hydroxyvitamin D 3 (25(OH)D 3 ) levels and exercise on physical fitness in community-dwelling frail elderly in Japan. A longitudinal survey was conducted in a town (latitude 36° north). Eighty women aged 65 years and over attended a 3-month exercise class. A face-to-face interview was conducted based on a questionnaire. The serum levels of 25(OH)D 3 , intact parathyroid hormone (iPTH), were measured. Nine physical fitness tests were performed at baseline and at the end of a 3-month follow-up period. Among 80 subjects, 56.3% experienced falls, and 71.3% experienced stumbling more than once during the past year. The prevalence of 25(OH)D 3 <50nmol/l or 25(OH)D 3 <75nmol/l was 27.5% and 88.8%, respectively. Significantly greater improvements in alternate step, functional reach (FR), “timed up & go” (TUG), and 5-m walk, and superior functional capacity for the subjects with 25(OH)D 3 levels greater than 67.5nmol/l (highest quartile) was observed at the end of the class. In contrast, the subjects with 25(OH)D 3 levels <47.5nmol/l (lowest quartile) did not improve their physical fitness. A serum 25(OH)D 3 level of greater than 47.5nmol/l may therefore be necessary to maintain walking ability and balance. Greater than 67.5nmol/l appears to be preferable for lower extremity strength in Japanese frail elderly women.