This study investigated the effect of μCT scanning with voxel sizes between 6–30 μm on mouse trabecular bone measures. Two commonly used segmentation methods for determining a global segmentation threshold were compared. Voxel size strongly affected trabecular bone parameters including connectivity density and trabecular thickness. The segmentation methods yielded similar outcomes for scans with small voxel sizes, but not scans with large voxel sizes. Suggest that high-resolution scans should be used when possible to accurately estimate trabecular bone microstructure.