Accurate measurements of knee joint motion during gait after anterior cruciate ligament reconstruction (ACLR), especially for the patellofemoral joint, are sparse. The aim of this study was to measure six-degree-of-freedom (6-DOF) patellofemoral and tibiofemoral motion in ACLR and uninjured contralateral knees during gait, and to compare these results to healthy (control) knees. Biplane fluoroscopy was used to measure 6-DOF patellofemoral and tibiofemoral motion in 15 ACLR participants (26.3 ± 3.9 years) for complete cycles of level walking and downhill walking, and the results were compared to data for 10 healthy individuals (29.8 ± 6.1 years). Mean patellar superior translation, anterior translation, and flexion over the gait cycle were respectively 4.4–5.6 mm greater, 5.4–6.3 mm greater, and 3.7°–7.0° less in the ACLR and contralateral knees compared to controls across both activities (p < 0.021). Articular contact was 7.6 mm higher on the femoral trochlea in the ACLR and contralateral knees compared to controls. The patellar tendon was 8.9 mm longer (p < 0.001) in the ACLR and contralateral knees compared to controls. Among ACLR participants, 14 out of 30 knees (47%) had an Insall–Salvati ratio ≥ 1.20, indicating patella alta. Mean tibial external rotation and anterior translation over the gait cycle were respectively 3.4°–3.8° greater and 2.6–3.0 mm greater in the ACLR knee compared to controls across both activities (p < 0.025). A high-riding patella in both knees of the ACLR participants was due to a longer patellar tendon. A change in the load-bearing areas of the femoral trochlea may contribute to the high rate of patellofemoral osteoarthritis seen after ACLR.