Abstract: Collagen IX is a fibril-associated collagen composed of three peptide chains encoded by three independent genes. The short form is expressed by osteoblasts while the long form is expressed by chondroblasts. We found that collagen IX-deficiency in mice by genetic inactivation of one col9a1 allele (col9a1+/-) developed osteoporosis. The osteoporotic bone structure manifested as mice aged with other associated phenotypes, including thoracic kyphosis and loss of body weight. Interestingly, despite reduced col9a1 synthesis in osteoblasts, mineralization in vitro and bone deposition demonstrated by vital labeling were not affected. Instead, osteoclasts in the col9a1+/- mice displayed striking flattened and enlarged cytoplasms potentially resorb more bones. A novel system testing bone substrate resorption by murine monoclonal osteoclast derived from M-CSF/RANKL-treated RAW264.7 was established and validated. When cultured on the col9a1+/- calvarial bone, these cells expanded over the bone substrate and formed large resorption pits. In silico studies identified non-coding homologous regions upstream and downstream 10kb of long and short COL9A1 between human and mouse sequence, and single nucleotide polymorphisms that potential have functional regulation were listed. Distinct bone microarchitecture of C57B1/6J, DBA/2J, and C3H/HeJ, as described concomitantly by using micro-computated tomography, indicated genetic determinants are involved in frail bone structure. C3H/HeJ osteoblasts displayed higher activities in vitro compared to C57B1/6J. Steady-state mRNA from calvarial and lumbar tissue showed more variable expression in collagen IX genes while other bone markers remained similar between the two strains. Congenic mice carrying DBA/2J allele on C57Bl/6J background were also characterized. The bone properties from congenic mice carrying col9a1 allele displayed similar tendency to their parental strains despite their variable collagen IX gene expressions. Functional genomics showed surprising complex molecular interaction that contributed to bone phenotypes. Together, these data provide collagen IX as a potential candidate gene in frail bone microarchitecture.