Background: An anterior cruciate ligament (ACL) tear impairs knee biomechanics in daily activities and potentially breaks the synergy among other knee ligaments. Previous studies have demonstrated that the biomechanics of collateral ligaments is influenced by ACL deficiency. Purpose: To investigate changes in the elongation patterns of the medial collateral ligament (MCL), lateral collateral ligament (LCL), and posterior cruciate ligament (PCL) during the execution of a single-leg squat before and after ACL reconstruction. Study Design: Descriptive laboratory study. Methods: A total of 16 patients (mean age, 24.9 ± 8.5 years) with ACL deficiency were enrolled in the study. Magnetic resonance imaging scans of the affected knees were used to produce 3-dimensional models of the tibia and femur and identify insertion sites of the MCL, LCL, and PCL. Motion capture of a single-leg squat was performed through a biplanar radiographic system. Data were acquired before ACL reconstruction and at a minimum of 18 months (mean, 22.9 ± 4.1 months) postoperatively. The centroids of the ligaments’ insertions were used to calculate the length of the investigated structures during the squat task. Absolute length ( L), absolute length increase from the orthostatic resting position (Δ L), and relative length increase (Δ L%) were computed for each ligament, and preoperative and postoperative data were compared using the paired Student t test. The intraclass correlation coefficient was used to determine the reliability of the ligament insertion's identification and kinematics between 2 independent observers. Results: Significant differences were found for the MCL in absolute length increase ( P = .047; Cohen d = 0.60) and relative length increase ( P = .043; Cohen d = 0.61) from rest between preoperatively and postoperatively (Δ Lpre = 1.0 mm; Δ Lpost = −1.1 mm; difference = 2.1 mm) at 0° to 30° of knee flexion during the descending phase of the single-leg squat. No differences were seen in the elongation patterns of the LCL or PCL from before to after ACL reconstruction. Conclusion: The MCL was significantly longer between 0° and 30° in ACL-deficient knees compared with ACL-reconstructed knees during the descending phase of a single-leg squat. No differences were identified for the LCL or PCL. Clinical Relevance: Early ACL reconstruction could have a protective effect on the MCL in combined ACL and MCL lesions.