Objective: Post-traumatic knee osteoarthritis (OA) is prevalent after anterior cruciate ligament reconstruction (ACLR). Biomarkers that identify individuals likely to develop OA, especially symptomatic OA, can help target preventative and therapeutic strategies. This study examined the magnitude and change over time in urinary CTX-II (uCTX-II) concentrations shortly after ACL reconstruction, and, secondarily, the associations with knee pain and function. Design: Subjects were 28 patients with ACLR and 28 age- and sex-matched controls (CNTRL). Testing was conducted at four time points spaced 4. weeks apart (4, 8, 12 and 16. weeks post-operative in ACLR). Measures included demographics, urine samples, Numeric Pain Rating Scale (NPRS) and International Knee Documentation Committee Subjective Knee Form (IKDC-SKF). uCTX-II concentrations were determined with competitive enzyme-linked immunosorbent assay (ELISA). uCTX-II concentrations at each time point in ACLR were compared to the mean concentration over time in CNTRL, with and without adjustment for body mass index (BMI). Changes over time in each measure and correlations between the slopes of change were examined. Results: uCTX-II concentrations were significantly higher in ACLR than CNTRL through 16 weeks post-operative when adjusted for BMI. In ACLR, uCTX-II concentrations significantly decreased over time, and the slope was associated with NPRS (r = 0.406, P = 0.039) and IKDC-SKF (r = -0.402, P = 0.034) slopes. Conclusion: uCTX-II concentrations shortly after ACLR were elevated compared to CNTRL and declined over time. Decreasing uCTX-II concentrations were associated with decreasing knee pain and improving function. uCTX-II may have a role as a prognostic marker following ACLR and warrants further investigation.