Maintenance of the walking ability is very important for smooth continuation of maintenance hemodialysis (HD). The aim of the present study was to clarify the physical activity level in daily living that HD patients should maintain to prevent deterioration of their walking ability. Outpatients undergoing maintenance HD, consisting of 65 males and 88 females with a mean age of 64 ± 11 years, were recruited for the present study. Their physical activity level was recorded over a week with an accelerometer. The physical activity level in daily living was defined as the sum of the lengths of time for which the patients were engaged in physical activity of light or greater intensity during the day, and expressed as the average duration per day. The walking ability was assessed by the normal walking speed and maximum walking speed. Data were analyzed using the receiver operating characteristic (ROC) curve, and the cut-off point for the physical activity time was determined to predict deterioration of the walking ability. In the prediction of deterioration of the normal and maximum walking speeds, the areas under the ROC curve for the physical activity time were 0.78 (95% confidence interval, 0.69–0.87, P < 0.001) and 0.75 (95% confidence interval, 0.63–0.86, P < 0.001), respectively. Moreover, the ROC curve revealed that the cut-off point for the physical activity time to prevent deterioration of the normal and maximum walking speeds was 50 min/day. Thus, HD patients should engage in physical activity for at least 50 min/day to prevent deterioration of their walking ability.