Although insulin-like growth factor-I (IGF-I) and estrogen signaling pathways have been shown to be involved in mediating the bone anabolic response to mechanical loading, it is not known whether these two signaling pathways crosstalk with each other in producing a skeletal response to mechanical loading. To test this, at 5 weeks of age, partial ovariectomy (pOVX) or a sham operation was performed on heterozygous IGF-I conditional knockout (H IGF-I KO) and control mice generated using a Cre-loxP approach. At 10 weeks of age, a 10 N axial load was applied on the right tibia of these mice for a period of 2 weeks and the left tibia was used as an internal non-non-loaded control. At the cortical site, partial estrogen loss reduced total volumetric bone mineral density (BMD) by 5%in control pOVX mice (P50.05, one-way ANOVA), but not in the H IGF-I KO pOVX mice. At the trabecular site, bone volume/total volume (BV/TV) was reduced by 5%-6%in both control pOVX (P,0.05) and H IGF-I KO pOVX (P50.05) mice. Two weeks of mechanical loading caused a 7%-8%and an 11%-13%(P,0.05 vs. non-loaded bones) increase in cortical BMD and cortical thickness (Ct.Th), respectively, in the control sham, control pOVX and H IGF-I KO sham groups. By contrast, the magnitude of cortical BMD (4%, P50.13) and Ct.Th (6%, P,0.05) responses were reduced by 50%in the H IGF-I KO pOVX mice compared to the other three groups. The interaction between genotype and estrogen deficiency on the mechanical loading-induced cortical bone response was significant (P,0.05) by two-way ANOVA. Two weeks of axial loading caused similar increases in trabecular BV/TV (13%-17%) and thickness (17%-23%) in all four groups of mice. In conclusion, partial loss of both estrogen and IGF-I significantly reduced cortical but not the trabecular bone response to mechanical loading, providing in vivo evidence of the above crosstalk in mediating the bone response to loading.