Muscle force of lower limb is a major factor for sustaining physical activity. Decreased muscle force can limit physical activity, which can increase mortality and morbidity in end-stage renal disease (ESRD) patients. Muscle force depends on several factors. One of the most important factors is 25-hydroxy vitamin D (25-OHD) that affects muscle function in both uremic and non-uremic patients. The aim of this study was to investigate the association between serum level of 25-OHD and muscle force of lower extremities in hemodialysis patients estimated by a Micro Manual Muscle Tester, a digital instrument that measures muscle force in kilograms This cross-sectional study was performed on 135 adult patients, 69 male (51%) and 66 female (69%) (mean: 1.4, standard deviation: 0.5), undergoing hemodialysis. Standard biochemistry parameters were measured before hemodialysis, including 25-OHD, calcium, albumin, para-hyroid hormone and C-reactive protein (CRP). Based on the result of serum level of 25-OHD, patients were classified into the following three groups: 85 patients (63%) were 25-OHD deficient (25-OHD <30), 43 patients (32%) had a normal level of 25-OHD (30-70) and seven patients (5%) had a toxic level of 25-OHD (>70) (mean: 1.42, standard deviation: 0.59). Also, based on the result of muscle force, patients were classified into the following three groups: 84/133 patients (62%) had weak muscle force (<5 kg), 46/133 patients (34%) had normal muscle force (5-10 kg) and three patients (21%) had strong muscle force (>10 kg) (mean: 1.39, standard deviation: 0.53). There was a significant relation between 25-OHD level and muscle force (P = 0.02), between age and muscle force (P = 0.002) and between gender and muscle force (P <0.001). In our opinion, 25-OHD can be a useful drug in ESRD patients to improve muscle force and physical activity.