Abstract Polymorphisms of the TNSALP gene have not previously been studied as a possible determinant for variations in BMD or as a predisposing genetic factor for osteoporosis. This study showed a significantly higher association between the 787T>C (Tyr246His) TNSALP gene and BMD among 501 postmenopausal women. Furthermore, the effects of amino acid substitution on the catalytic property of the protein translated from the 787T>C gene were examined. Introduction: Alkaline phosphatase (ALP) is present mainly on the cell membrane in various tissues and hydrolyzes a variety of monophosphate esters into inorganic phosphoric acid and alcohol. Human ALPs are classified into four types: tissue-nonspecific, intestinal, placental, and germ cell types. Based on studies of hypophosphatasia, which is a systemic skeletal disorder resulting from a tissue-nonspecific ALP (TNSALP) deficiency, TNSALP was suggested to be indispensable for bone mineralization. Materials and Methods: We explored the possibility that the TNSALP gene may contribute to age-related bone loss in humans by examining the association between TNSALP gene polymorphisms and BMD in 501 Japanese postmenopausal women. To analyze the protein translated from the TNSALP gene associated with BMD, we constructed a TNSALP cDNA expression plasmid. Results: We genotyped two single nucleotide polymorphisms (787T>C[Tyr246His] and 876A>G[Pro275Pro]), which proved to be in complete linkage disequilibrium. There was a significant difference in BMD and the BMD score adjusted for age and body weight (Z score) among haplotypes (p = 0.041), which was lowest among 787T/876A homozygotes, highest among 787T>C/876A>G homozygotes, and intermediate among heterozygotes. In subgroups divided by age, haplotypes were significantly associated with BMD in older postmenopausal women (>74 years; p = 0.001), but not in younger postmenopausal women (<74 years; p = 0.964). Expression of the 787T>C TNSALP gene using COS-1 cells showed that the protein translated from 787T>C had ALP-specific activity similar to that of 787T. Interestingly, the Km value for TNSALP in cells transfected with the 787T>C TNSALP gene was decreased significantly compared with that of cells bearing the 787T gene, reflecting the higher affinity. Conclusions: These results suggest that variation in TNSALP may be an important determinant of age-related bone loss in humans and that the phosphate metabolism pathway may provide a novel target for the prevention and treatment of osteoporosis.