Resumen: Hamstring muscle injuries are one of most frequent injuries in team sports. Whole-body vibration (WBV) has an important effect on flexibility that could prevent shortening of the hamstrings. To investigate both acute and residual effect of a single bout of WBV on hamstring flexibility in a group of university athletes from team sports 70 athletes (81% men, age 21 ± 1.9 years old) were separated into three groups; control group (CG; n = 24), hamstring flexibility group without vibration (-V; n = 23), and hamstring flexibility group with vibration (+V; n = 23). Both -V and +V groups performed the same experimental protocol, composed of 6 sets of 30 seconds of passive hamstring flexibility over a vibration platform with both legs alternately (full-length 6 minutes; 3 minutes per leg). A high-magnitude vibration loading was applied only in +V group (40 Hz and 4 mm). Hamstring flexibility was evaluated through the Modified Sit and Reach (MSR) and Passive Straight Leg Raise (PSLR) test before (baseline), immediately after (acute effect), and after 72 h (residual effect) intervention. Both experimental groups showed a significant improvement in flexibility compared to CG in all measures (p < 0.05). No statistical differences were found between +V and -V, however, MSR, right PSLR, and left PSLR residual effect size (Cohen's d) were greater in +V. In conclusion, adding a WBV stimulus to flexibility training improves acute and residual hamstring flexibility in university athletes from team sports.