No consensus has been reached on the serum 25-hydroxyvitamin D [25(OH)D] levels required to ensure optimal bone health around menarche. We searched for a possible interaction of 25(OH)D levels and calcium intake on lumbar spine mineralization and on biologic features of bone metabolism in healthy late-pubertal girls. Lumbar spine parameters (ie, area, mineral content, and density) and calcium intake were evaluated in 211 healthy white adolescent girls at pubertal stages IV-V (11 to 16.9 years), together with biologic markers of calcium and bone metabolism and with International External Quality Assessment Scheme for Vitamin D Metabolite (DEQAS)-validated serum 25(OH)D levels. A high prevalence of 25(OH)D levels 30 nmol/L (41%), 40 nmol/L (61%), and 50 nmol/L (70%) was found during winter-spring. Parathyroid hormone (PTH) levels were inversely associated with 25(OH)D levels ( p ¼ .0021). In contrast, lumbar spine mineral content and density were not associated with 25(OH)D, excepted when calcium intake was below 600 mg/day ( p ¼ .0081). Girls with such low calcium intake and 25(OH)D levels of 40 nmol/L or less (9% of the cohort) had a 0.4 to 0.7 SD lower mean areal bone mineral density Z-score than girls with higher calcium intake and/or higher 25(OH)D status. The adverse association between lumbar spine mineralization and combined calcium deficiency-low 25(OH)D levels remained significant in the 91 girls who could be followed over 4 years after their initial evaluation. We conclude that low 25(OH)D levels ( 40 nmol/L) are observed frequently during winterspring in late-pubertal European girls, which may exacerbate the negative impact of calcium deficiency on lumbar spine mineralization. ß