Osteocalcin is one of the most abundant noncollagenous proteins in bone. Phenotypes of osteocalcin knock-out mice (OC−/−) may vary on different backgrounds and with sex. Previous studies using adult female (OC−/−) mice on a mixed genetic background (129/B6) showed osteocalcin inhibited bone formation leading to weaker bone in wild-type (OC+/+). Yet on a pure (B6) genetic background male mice revealed osteocalcin improved fracture resistance and OC−/− bones were more prone to fracture. Osteocalcin is decreased with age and in some diseases (diabetes) where bone weakness is observed. The effect of osteocalcin in adult female bone from mice on a pure B6 background is unknown. We investigated differences in bone mineral properties and bone strength in female adult (6 months) (OC+/+) and (OC−/−) mice on a pure C57BL/6J background using Fourier Transform Infrared Imaging (FTIRI), micro-computed tomography (uCT), biomechanical measurements, histomorphometry and serum turnover markers (P1NP, CTX). Similar to female age matched mice on the (129/C57) background we found B6 OC−/− mice had a higher bone formation rate, no change in bone resorption, more immature mineral, decreased crystallinity and increased trabecular bone as compared to OC+/+. In contrast, the OC−/− mice on a pure B6 background had a lower bone mineral density, lower mineral to matrix ratio resulting in reduced stiffness and weaker bone strength. Our results demonstrate some properties of the OC−/− phenotype are dependent on genetic background. This may suggest that reduced osteocalcin may contribute to fracture and weaker bone in some groups of elderly and adults with diseases where osteocalcin is low. •Female Osteocalcin knock-out mice (OC-/-) on pure B6 background had increased bone formation vs. OC+/+.•OC-/- bones had immature mineral phase, increased carbonate in mineral and decreased crystallinity as vs. OC+/+.•Contrary to OC-/- on 129/B6 background, a decreased mineral/matrix ratio in OC-/- cortical vs OC+/+ bones on B6 background.•Contrary to OC-/- on 129/B6 background, reduced stiffness and strength in OC-/- vs. OC+/+ bone on B6 background.•Some mineral properties are dependent on genetic background in OC-/- mice.