Objective To examine the acute effects of aquatic and land treadmill exercise on gait kinematics as well as the level of disease-specific and movement-related pain for individuals with osteoarthritis. Design Quasi-experimental crossover design. Setting Biomechanics laboratory. Participants Participants (N=14; age, 43–64y) diagnosed with osteoarthritis at the knee (n=12), osteoarthritis at the knee and ankle (n=1), or osteoarthritis at the knee and hip (n=1). Interventions Participants performed 3 exercise sessions separated by at least 24 hours in 1 week for each mode of exercise (aquatic treadmill and land treadmill). Main Outcome Measures Gait kinematics and pain were measured before and after each intervention. Results The angular velocity gain score during stance for left knee extension was improved by 38% after aquatic treadmill exercise (P=.004). Similarly, during swing, the gain scores for angular velocity were also greater for left knee internal rotation and extension by 65% and 20%, respectively (P=.004, P=.008, respectively). During stance, the joint angle gain score for left hip flexion was 7.23% greater after land exercise (P=.007). During swing, the angular velocity gain score for right hip extension was significantly greater for aquatic exercise by 28% (P=.01). Only the joint angle gain score for left ankle abduction during stance was significantly higher after land exercise (4.72%, P=.003). No other joint angle gain scores for either stance or swing were significantly different for either condition (P=.06–.96). Perceived pain was 100% greater after land than aquatic treadmill exercise (P=.02). Step rate and step length were not different between conditions (P=.31–.92). Conclusions An acute training period on an aquatic treadmill positively influenced joint angular velocity and arthritis-related joint pain. Acute aquatic treadmill exercise may be useful as a conservative treatment to improve angular speed of the lower-extremity joints and pain related to osteoarthritis.