The prevalence of osteoporosis is high among postmenopausal women and individuals sustaining a spinal cord injury (SCI). We assessed the effects of estrogen loss and unloading on the trabecular bone of the knee in women. Pre- and postmenopausal ambulatory women (n=17) were compared to pre- and postmenopausal women with SCI (n=20). High-resolution magnetic resonance imaging was used to compare groups on apparent measures of trabecular bone volume, trabecular number, trabecular spacing, and trabecular thickness in the distal femur and proximal tibia, regions with a high proportion of trabecular bone and the most common fracture site for SCI patients. Trabecular bone was deteriorated in women with SCI compared to ambulatory women. SCI groups had fewer, (-19 and -26% less) and thinner trabeculae (-6%) that were spaced further apart (40% and 62% more space between structures) resulting in less trabecular bone volume (-22% and -33%) compared to the ambulatory groups (tibia and femur, respectively). Postmenopausal women with SCI also had 34% greater trabecular spacing in the tibia compared to the 40-year-old premenopausal women with SCI, showing an interaction between unloading and estrogen loss. Middle-aged postmenopausal, ambulatory women, not taking estrogen or medications that affect bone, did not show the deteriorated trabeculae that were evident in women with SCI, nor did they show differences in distal femur and proximal tibia trabeculae compared to a premenopausal group. We conclude that the effect of unloading on bone architecture is greater than that of estrogen loss in middle-aged women.