Peak eccentric force during the Nordic hamstring exercise (NHE) is recognized as a predictive factor for hamstring strain injury (HSI). During the NHE, the knee flexor muscles are eccentrically contracting to resist the knee joint extension. Therefore, it is thought that the action of the gastrocnemius muscle, and thus the ankle position, influences peak eccentric force during the NHE. However, the effect of ankle position on peak eccentric force during the NHE remains unclear. Therefore, we investigated the effect of ankle position on peak eccentric force during the NHE in a cohort of 50 healthy young male rugby players (mean age, 18.7 ± 1.2 years; mean body mass, 81.7 ± 15.2 kg; height, 1.72 ± 0.06 m) with no history of HSI. Each participant performed NHE strength testing with the ankle dorsiflexed or plantarflexed position and was instructed to fall forward as far as possible within 3 s. Peak eccentric force, reported relative to body mass (N/kg), of both legs was recorded, and the mean values of both legs were compared in both ankle positions. The mean peak eccentric force was significantly greater with the ankle plantarflexed position than the dorsiflexed position (3.8 ± 1.1 vs. 3.5 ± 1.1 N/kg, respectively, p = 0.049). These results indicate that ankle position should be carefully considered when measuring peak eccentric force during the NHE and performing NHE training.