Objectives: Reduced weight-loading on the lower extremities during growth induces abnormal bone morphology and malalignment, but the effects of reloading on these abnormalities remain unknown. We aimed to determine the specific timeframes in which hindlimb suspension (HS) during growth induces abnormal femoral morphologies and knee malalignments present at skeletal maturity in rats. Methods: Four-week-old female rats were subjected to HS for one, two, or four weeks using the tail suspension method, followed by reloading. Age-matched rats were used as controls. At 12 weeks of age, femoral morphology and knee joint alignment were assessed using X-ray computed tomography. Results: Femoral anteversion angle, medial/lateral condylar height ratio (MC/LC), and patellar tilt angle were significantly larger, while trochlear angle, lateral condylar height (LC), and lateral trochlear inclination (LTI) were smaller in the groups that underwent HS for two or four weeks prior to reloading, compared with the control group. There were significant correlations between LTI and LC (r = 0.57), MC/LC (r = -0.42), and patellar tilt angle (r = -0.49). In the 4-week HS group, lateral patellar dislocation was observed in 3 of 8 animals. Conclusion: Insufficient weight-loading more than two weeks during growth may induce knee joint instability even after reloading.