Context: There are numerous studies on the benefits of dry needling (DN) for pain relief. No studies exist examining the effects of DN on hamstring flexibility. Objective: To determine the immediate effects of DN on hamstring flexibility in healthy subjects with shortened hamstrings. Design: A single-blinded, pretest–posttest clinical pilot study. Setting: A university physiotherapy clinic. Subjects: A total of 15 healthy subjects (female = 11; age = 23.26 [4.3] y) with shortened hamstrings participated in this study. Intervention: Subjects received a single session of DN. Three locations on the hamstring muscle group were needled, each for 1 minute. Main Outcome Measures: The active knee extension test, muscle compliance, passive peak torque, and stretch tolerance were measured at baseline, immediately, and 15 minutes after DN. Results: There were statistically significant improvements in all outcome measures immediately after DN and at the 15-minute follow-up. The effect sizes for all outcome measures were large (Cohen’s d ≥ 0.8). No serious adverse events were observed with DN. Conclusions: This is the first study that demonstrates the beneficial effects of DN on hamstring flexibility, muscle compliance, and stretch tolerance without added stretching. The beneficial effects of DN should encourage clinicians to use DN as a novel strategy for increasing muscle flexibility.