Background Jack-knife stretching is a newly developed static hamstring stretching exercise. However, no studies have compared this stretching exercise to hamstring stretching with proprioceptive neuromuscular facilitation (PNF). Purpose This study compared the effects of PNF hamstring stretching and jack-knife stretching on hamstring flexibility. Study design Randomized controlled clinical trial Methods Twenty-four adults with bilateral hamstring tightness were randomly assigned to the PNF hamstring stretching or jack-knife stretching group. All participants performed PNF hamstring stretching or jack-knife stretching for 150 s. Before and after hamstring stretching, the participants completed the active knee extension (AKE), active straight leg raise (ASLR), and finger-to-floor distance (FFD) tests. Lumbar flexion and the anterior pelvic tilt angle were measured during the FFD test. Results Both hamstring stretching exercises increased the knee extension angle during the AKE test, hip flexion angle during the ASLR test, and lumbar flexion and anterior pelvic tilt angle during the FFD test while decreasing the FFD value (<italic>p</italic><0.05). However, no significant between-group differences were found (<italic>p</italic>>0.05). Conclusions PNF hamstring stretching and jack-knife stretching have similar beneficial effects on hamstring flexibility.