The relative contributions of sex differences in anatomy, biomechanics, and hormones to the increased risk of anterior cruciate ligament (ACL) injury in female athletes remains unknown. The purpose of this study is to investigate sex differences in anatomy and biomechanics of the native and reconstructed ACL using our established murine model. A total of 140 12-week-old wild-type C57Bl/6 (70 male vs. 70 female) mice were used for this study. ACL reconstruction was performed on 120 mice who were split into four groups:​ Group 1 (30 males sacrificed at 14 days), Group 2 (30 females sacrificed at 14 days), Group 3 (30 males sacrificed at 28 days), and Group 4 (30 females sacrificed at 28 days). Tendon graft-to-bone healing was assessed by biomechanical, histological, and micro-CT analysis. Twenty mice were used for baseline testing. Females showed significantly higher anterior (p < 0.05) and total displacement (p < 0.05). Males demonstrated a significantly higher load-to-failure force of native ACLs compared to females (p < 0.05). There was no significant difference in load-to-failure force in the ACL autograft. There were no significant sex differences in histological analysis of graft integration or tibial slope. The increased knee laxity and reduced load-to-failure of the native ACL observed in the female mice are consistent with some of the proposed risk factors driving the increased risk of ACL injury in females. Understanding the relative contributions of factors driving sex differences in material properties of the ACL will provide insight into the sex differences in ACL injury and future prevention strategies.