Background. The aim of this study was to gain a deeper understanding of the gender differences in knee osteoarthritis (OA) by evaluating the differences in gait spatio-temporal parameters and the differences in pain, quality of life and function between males and females suffering from knee OA. Methods. 49 males and 85 females suffering from bilateral medial compartment knee OA participated in this study. Each patient underwent a computerized gait test and completed the WOMAC questionnaire and the SF-36 health survey. Independent t-tests were performed to examine the differences between males and females in age, BMI, spatio-temporal parameters, the WOMAC questionnaire and the SF-36 health survey. Results. Males and females had different gait patterns. Although males and females walked at the same walking speed, cadence and step length, they presented significant differences in the gait cycle phases. Males walked with a smaller stance and double limb support, and with a larger swing and single limb support compared to females. In addition, males walked with a greater toe out angle compared to females. While significant differences were not found in the WOMAC subscales, females consistently reported higher levels of pain and disability. Conclusion. The spatio-temporal differences between genders may suggest underlying differences in the gait strategies adopted by males and females in order to reduce pain and cope with the loads acting on their affected joints, two key aspects of knee OA. These gender effects should therefore be taken into consideration when evaluating patients with knee OA. Trial Registration. The study is registered in the NIH clinical trial registration, protocol No. NCT00599729.