Osteoporosis is a common disease with a strong genetic component. We previously described two polymorphic sites in the COL1A1 gene promoter, -1997 G/T and -1663indelT, which have been associated with bone mineral density (BMD), a surrogate trait for osteoporosis. Here, we explore the molecular mechanisms underlying this association by performing transient transfections in MG-63 cells of constructs bearing different COL1A1 promoter regions, containing different alleles or haplotypes of the polymorphic sites. These promoter regions drove the transcription of a luciferase reporter gene. The main differences in transcriptional activity relied on an inhibitory region localized to the -1284 to -254 interval. Regarding the polymorphisms, reproducible differences were observed between the alleles of each of them: the G allele at -1997 showed a higher transcriptional activity than the T allele, as did the 7T allele of -1663 as compared with 8T. Accordingly, the T-8T haplotype was the weakest transcriber. A functional interaction was found between the -1997 and -1663 polymorphisms, in that the difference in transcriptional activity between the 7T and 8T alleles was dependent on the allele at -1997. This different transcriptional activity of the two -1663indelT alleles correlated with different binding capacities of the corresponding oligonucleotides to osteoblast nuclear proteins. Supershift assays allowed us to identify one of these proteins as the architectural transcription factor Nmp4/CIZ, a protein known to be an inhibitor of BMP/Smad signalling.