Type III Collagen (Col3), a fibril-forming collagen, is a major extracellular matrix component in a variety of internal organs and skin. It is also expressed at high levels during embryonic skeletal development and is expressed by osteoblasts in mature bone. Loss of function mutations in the gene encoding Col3 (Col3a1) are associated with vascular Ehlers Danlos Syndrome (EDS). Although the most significant clinical consequences of this syndrome are associated with catastrophic failure and impaired healing of soft tissue structures, several studies have documented skeletal abnormalities in vascular EDS patients. However, there are no reports of the role of Col3 deficiency on the murine skeleton. We compared craniofacial and skeletal phenotypes in young (6-8 weeks) and middle-aged (>1 year) control (Col3+/+) and haploinsufficient (Col3+/−) mice, as well as young null (Col3−/−) mice using microcomputed tomography (μCT). Although Col3+/− mice did not have significant craniofacial abnormalities based upon cranial morphometrics, microCT analysis of distal femur trabecular bone demonstrated significant reductions in bone volume (BV), bone fraction volume (BV/TV), connectivity density (ConnD), structure model index (SMI) and trabecular thickness (TbTh) in young adult, female Col3+/− mice relative to wild-type littermates. The reduction in BV/TV persisted in female mice at one year of age. Next we evaluated the role of Col3 in vitro. Osteogenesis assays revealed that cultures of mesenchymal progenitors harvested from Col3−/− embryos display decreased alkaline phosphatase activity and reduced capacity to undergo mineralization. Consistent with this data, a reduction in osteogenic markers (type I collagen, osteocalcin and bone sialoprotein) correlates with reduced bone Col3 expression in Col+/− mice and with age in vivo. A small but significant reduction in osteoclast numbers was found in Col3+/− compared to Col3+/+ bones. Taken together, these findings indicate that Col3 plays a role in development of trabecular bone through its effects on osteoblast differentiation.