Insulin-like growth factor (IGF) I is a critical peptide for skeletal growth and consolidation. However, its regulation is complex and, in part, heritable. We previously indicated that changes in both serum and skeletal IGF-I were related to strain-specific differences in total femoral bone mineral density (BMD) in mice. In addition, we defined four quantitative trait loci (QTLs) that contribute to the heritable determinants of the serum IGF-I phenotype in F2 mice derived from progenitor crosses between C3H/HeJ (C3H; high total femoral BMD and high IGF-I) and C57BL/6J (B6; low total femoral BMD and low IGF-I) strains. The strongest QTL, IGF-I serum level 1 (Igflsl-1; log10 of the odds ratio [LOD] score, approximately 9.0), is located on the middle portion of chromosome (Chr) 6. For this locus, C3H alleles are associated with a significant reduction in serum IGF-I. To test the effect of this QTL in vivo, we generated a new congenic strain (B6.C3H-6T [6T]) by placing the Chr 6 QTL region (D6Mit93 to D6Mit150) from C3H onto the B6 background. We then compared serum and skeletal IGF-I levels, body weight, and several skeletal phenotypes from the N9 generation of 6T congenic mice against B6 control mice. Female 6T congenic mice had 11-21% lower serum IGF-I levels at 6, 8, and 16 weeks of age compared with B6 (p < 0.05 for all). In males, serum IGF-I levels were similar in 6T congenics and B6 controls at 6 weeks and 8 weeks but were lower in 6T congenic mice at 16 weeks (p < 0.02). In vitro, there was a 40% reduction in secreted IGF-I in the conditioned media (CMs) from 6T calvaria osteoblasts compared with B6 cells (p < 0.01). Total femoral BMD as measured by peripheral quantitative computed tomography (pQCT) was lower in both 6T male (-4.8%, p < 0.01) and 6T female (-2.3%, p = 0.06) congenic mice. Geometric features of middiaphyseal cortical bone were reduced in 6T congenic mice compared with control mice. Femoral cancellous bone volume (BV) density and trabecular number (Tb.N) were 50% lower, whereas trabecular separation (Tb.Sp) was 90% higher in 8-week-old female 6T congenic mice compared with B6 control mice (p < 0.01 for all). Similarly, vertebral cancellous BV density and Tb.N were lower (-29% and -19%, respectively), whereas Tb.Sp was higher (+29%) in 16-week-old female 6T congenic mice compared with B6 control mice (p < 0.001 for all). Histomorphometric evaluation of the proximal tibia indicated that 6T congenics had reduced BV fraction, labeled surface, and bone formation rates compared with B6 congenic mice. In summary, we have developed a new congenic mouse strain that confirms the Chr 6 QTL as a major genetic regulatory determinant for serum IGF-I. This locus also influences bone density and morphology, with more dramatic effects in cancellous bone than in cortical bone.