Anterior cruciate ligament (ACL) rupture alters knee kinematics and contributes to premature development of osteoarthritis. However, there is limited data regarding the in vivo biomechanical response of tibiofemoral cartilage to activities of daily living (ADLs) in ACL-deficient knees. In this study, eight otherwise healthy participants with chronic unilateral ACL deficiency completed a stress test to assess the effect of 20 min of level treadmill walking at a speed of 2.5 mph on tibiofemoral cartilage in their ACL-deficient and contralateral ACL-intact knees. Three-dimensional surface models developed from pre- and post-activity magnetic resonance (MR) images of the injured and uninjured knees were used to determine compressive strain across multiple regions of tibiofemoral cartilage (medial and lateral tibial plateaus, medial and lateral femoral condyles, medial aspect of femoral condyle adjacent to intercondylar notch of the femur). In the ACL-deficient knees, we observed significantly increased cartilage strain in the region of the medial femoral condyle adjacent to the intercondylar notch (6% in deficient vs. 2% in contralateral, p = 0.01) as well as across the medial and lateral tibial plateaus (4% vs. 3%, p = 0.01) relative to the contralateral ACL-intact knees. Increased compressive strain at the medial intercondylar notch and tibial plateau suggests alterations in mechanical loading or the response to load in these regions, presumably related to altered knee kinematics. These changes may disrupt cartilage homeostasis and contribute to subsequent development of osteoarthritis.