Objective: We evaluated associations between vitamin D status and skeletal muscle, strength, and bone mineral density (BMD) outcomes after ACL reconstruction (ACLR) in an observational study. Methods: Serum measures included 25-hydroxyvitamin D (25(OH)D; free and total), vitamin D binding protein (DBP), and 1,25-dihydroxy vitamin D (1,25(OH)2D) at baseline, 1 week, 4 months, and 6 months post-ACLR. Vastus lateralis biopsies were collected from the healthy and ACL-injured limb of 21 young, healthy participants (62% female; 17.8 [3.2] yr, BMI: 26.0 [3.5] kg/m2) during ACLR and the injured limb only at 1 week and 4 month follow ups. RNA and protein were isolated from biopsies and assessed for vitamin D receptor [VDR], and vitamin D-activating enzymes. Quadriceps fiber cross-sectional area (CSA) was determined with immunohistochemistry. BMD of femur and tibia were determined with at baseline and 6 months post-ACLR; strength was assessed with an isokinetic dynamometer. Results: 1,25(OH)2D decreased from baseline to one week after ACLR (21.6 [7.9] vs. 13.8 [5.5] pg/mL; p<0.0001). VDR and 25-hydroxylase transcript abundance and VDR and DBP proteins were elevated one week after ACLR compared with baseline (FDR<0.05; p<0.05). Participants with an average total 25(OH)D <30 ng/mL showed significant decreases in CSA 1 week and 4 months after ACLR (p<0.01; p=0.041 for time x D status interaction), whereas those with total 25(OH)D ≥30ng/mL showed no significant differences (p>0.05 for all comparisons). BMD and strength measures were lower at follow up but did not associate with vitamin D status. Conclusion: ACLR promotes vitamin D pathways in the quadriceps and low status is associated with loss of skeletal muscle both 1 week and 4 months after ACLR.