Non-muscular tissue stiffness is assumed to have a negative impact on joint flexibility, and a reduction in non-muscular tissue stiffness may be important, especially in older adults. The present study aimed to compare the acute effects of static stretching on non-muscular tissue stiffness between older and young adults and to investigate whether a decrease in tissue stiffness improves joint flexibility. Twenty older (62-83years) and 20 young (21-24years) males participated. Ankle dorsiflexion static stretching (five sets of 90s each) was performed, and before and after stretching, the ankle dorsiflexion range of motion (RoM), passive ankle joint stiffness, and shear wave speed (SWS) (an index of stiffness) of the sciatic nerve, tibial nerve, and posterior thigh fascia were measured. Stretching led to an increase in RoM and a decrease in passive joint stiffness in both groups (P < 0.001) with no significant between-group differences (P ≥ 0.055). The between-group difference in the effect of stretching on SWS was evident only for the sciatic nerve, and a decline in sciatic nerve SWS was only observed in the older adult group (pre-stretching: 2.5 ± 0.3m/s; post-stretching: 2.3 ± 0.4m/s; P = 0.027). A significant positive repeated-measures correlation was observed between the sciatic nerve SWS and passive joint stiffness (P = 0.014, rrm = 0.540). The reduction in sciatic nerve stiffness by stretching was noticeable in older men and led to improved joint flexibility. These findings may provide insight into tissue adaptation by stretching and may be used to explore effective exercises for improving joint flexibility in older adults.