Nordic Hamstring Exercise (NHE) is one of the best exercises proposed for injury prevention of hamstring muscles. However, its effects on lower extremity proprioception are unclear. The aim of this study was to investigate the immediate effects of a single bout of NHE on hip and knee joints’ proprioception. Forty collegiate male soccer players participated in this study with a mean age of 22.85 ± 1.82 years and were randomized into either control (n = 20) or experimental (n = 20) groups. Each subject participated in pre-test measurements in which hip and knee active joints position sense (JPS) were assessed in standing and lying tasks using the image-capturing method. The experimental group then performed three sets of NHE with 10 repetitions in each set, while the control group rested for 10 min. Paired and independent t-tests were used for calculating the differences within and between groups on SPSS software, respectively. The level of significance was P ≤ 0.05. Hip JPS in the lying task and knee JPS in both of the standing and lying tasks were impaired significantly after performing a single bout of NHE (P ≤ 0.05). However, the effects of this exercise on hip JPS in the standing task were not significant (P ≥ 0.05). NHE performing with three sets of 10 repetitions can significantly impair hip and knee JPS immediately after exercise and reduce the proprioception acuity of the lower limbs. It is recommended to perform this exercise at a time rather than before training or match sessions.