Biomechanical factors (e.g., joint loading) have a significant role in the progression of osteoarthritis (OA). However, some relationships between in vivo joint loading indices and tibial cartilage thickness are conflicting. This study investigated relationships between pre‐operative in vivo external knee joint moments, joint alignment and regional tibial cartilage thickness using micro‐CT in subjects with end‐stage knee OA. Tibial plateaus from 25 patients that underwent knee replacement for OA were micro‐CT scanned (17 µm/voxel). Prior to surgery, subjects underwent gait analysis to calculate external knee moments. The mechanical axis deviation (MAD) was obtained from pre‐operative radiographs. Cartilage thickness (Cart.Th) was analyzed from micro‐CT images, in anteromedial, anterolateral, posteromedial and posterolateral subregions of interest. Medial‐to‐lateral Cart.Th ratios were also explored. Relationships between Cart.Th and joint loading indices were examined using Pearson's correlations. Significant correlations were found between Cart.Th and joint loading indices, positive anteromedially with the first peak knee adduction moment (r = 0.55, p < 0.01) and external rotation moment (ERM; r = 0.52, p < 0.01), and negative with MAD (r = −0.76, p < 0.001). In the lateral regions, these correlations had opposite signs. The medial‐to‐lateral Cart.Th ratio correlated strongly with ERM (r = 0.63, p = 0.001) and MAD (r = −0.75, p < 0.001). Joint loading indices correlated with regional cartilage thickness values and their medial‐to‐lateral ratios in end‐stage knee OA subjects, with higher regional loads corresponding to thinner cartilage. These relationships have the opposite sign compared to the subchondral bone microarchitecture found in our previous study on the same specimens, which may suggest a complementary bone–cartilage interplay in response to loading.