Aberrant joint loading contributes to the development of posttraumatic knee osteoarthritis (PTOA) following anterior cruciate ligament reconstruction (ACLR);​ yet little is known about the association between joint loading due to daily walking and cartilage health post-ACLR. Accelerometer-based measures of daily steps and cadence (i.e., rate of steps/min) provide information regarding daily walking in a real-world setting. The purpose of this study was to determine the association between changes in serum cartilage oligomeric matrix protein (COMP;​ %∆COMP), a mechanosensitive biomarker that is associated with osteoarthritis progression, following a standardized walking protocol and daily walking in individuals with ACLR and uninjured controls. Daily walking was assessed over 7 days using an accelerometer worn on the right hip in 31 individuals with ACLR and 21 controls and quantified as mean steps/day and time spent in ≥100 steps/min. Serum COMP was measured before and following a 3000-step walking protocol at a preferred speed. %∆COMP was calculated as a change in COMP relative to the prewalking value. Linear regressions were used to examine associations between daily walking and %∆COMP after adjusting for preferred speed. Fewer daily steps (ΔR² = 0.18, p = 0.02) and fewer minutes spent in ≥100 steps/min (ΔR² = 0.16, p = 0.03) were associated with greater %∆COMP following walking in individuals with ACLR;​ no statistically significant associations existed in controls (daily steps:​ ΔR² = 0.03, p = 0.47;​ time ≥100 steps/min:​ ΔR² < 0.01, p = 0.81). Clinical significance:​ Individuals with ACLR who engage in less daily walking undergo greater %ΔCOMP, which may represent greater cartilage degradation or turnover in response to walking.