Most cases of unilateral knee osteoarthritis (OA) progress to bilateral OA within 10 years. Biomechanical asymmetries have been implicated in contralateral OA development; however, gait analysis alone does not consistently detect asymmetries in OA patient gait. Stair ambulation is a more demanding activity that may be more suited to reveal between-leg asymmetries in OA patients. The objective of this study was to investigate the between-leg biomechanical differences in patients with unilateral mild-to-moderate knee OA. Sixteen unilateral mild-to-moderate medial knee OA patients and 16 healthy individuals underwent kinematic and kinetic analysis of stair ascent and descent. Stair ascent produced higher loading and muscle forces in the unaffected limb compared to the OA limb, and stair descent produced lower loading on the OA limb compared to healthy subjects. These biomechanical differences were apparent in the ankle, knee, and hip joints. The implications of these findings are that OA patients rely more heavily on their unaffected sides than the affected side in stair ascent, a strategy that may be detrimental to the unaffected joint health. The reduction in affected limb loading in stair descent is thought to be related to minimizing pain.