Objective The purpose of this study was to measure serum 25-hydroxyvitamin D [25(OH)D] levels in Japanese patients with diabetic nephropathy and determine the relationship between 25(OH)D concentrations and various factors. Methods The study subjects included 442 patients with type 2 diabetes. Their serum levels of creatinine, HbA1c, intact-parathyroid hormone, urinary albumin, 25(OH)D, and 1,25-dihydroxyvitamin D [1,25(OH)2D] were measured and their estimated glomerular filtration rate (eGFR) was determined. The patients were divided into four groups based on the risk for progression to chronic kidney disease (CKD): low, moderate, high, very high, based on their eGFR and their level of albuminuria. Results The median 25(OH)D level was 14.6 ng/mL; 11% of the patients had 25(OH)D deficiency (<10 ng/ mL), and 2% of patients had active vitamin D deficiency, as defined by a 1,25(OH)2D level of <22 pg/mL. The serum 25(OH)D level was correlated with the serum 1,25(OH)2D level in patients with a very high risk for CKD, but not in those with a moderate or high risk for CKD. Conclusion Although the vitamin D levels of the Japanese patients with diabetic nephropathy and CKD were low, the prevalence of vitamin D deficiency, as defined by the 1,25(OH)2D level, was low. Albuminuria, younger age, and female gender were associated with a low 25(OH)D level. The serum level of 25(OH)D should be monitored to assess the vitamin D status of patients with nephropathy and CKD.