Anterior cruciate ligament (ACL) deficient patients have an increased rate of patellofemoral joint (PFJ) osteoarthritis (OA) as compared to the general population. Although the cause of post-injury OA is multi-factorial, alterations in joint biomechanics may predispose patients to cartilage degeneration. This study aimed to compare in vivo PFJ morphology and mechanics between ACL deficient and intact knees in subjects with unilateral ACL ruptures. Eight male subjects underwent baseline MRI scans of both knees. They then performed a series of 60 single-legged hops, followed by a post-exercise MRI scan. This process was repeated for the contralateral knee. The MR images were converted into three-dimensional surface models of cartilage and bone in order to assess cartilage thickness distributions and strain following exercise. Prior to exercise, patellar cartilage was significantly thicker in intact knees as compared to ACL deficient knees by 1.8%. In response to exercise, we observed average patellar cartilage strains of 5.4 ± 1.1% and 2.5 ± 1.4% in the ACL deficient and intact knees, respectively. Importantly, the magnitude of patellar cartilage strain in the ACL deficient knees was significantly higher than in the intact knees. However, while trochlear cartilage experienced a mean strain of 2.4 ± 1.6%, there was no difference in trochlear cartilage strain between the ACL deficient and uninjured knees. In summary, we found that ACL deficiency was associated with decreased patellar cartilage thickness and increased exercise-induced patellar cartilage strain when compared to the uninjured contralateral knees.