In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K (10 nM) and 1α,25-dihydroxyvitamin D (1,25(OH)D) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (+/+) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K (10 nM) and 1,25(OH)D (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K and 1,25(OH)D co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K plus 1,25(OH)D treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K plus D combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis.