The purpose of the present study was to examine the effect of Yang-style Tai chi (TC) on gait parameters and musculoskeletal flexibility in healthy Chinese female adults. Sixty-six female adults aged >65 years were randomly assigned to either an experimental group (67.9 ± 3.2 years of age) receiving three 90-min simplified 24-form TC sessions for eight weeks, or a control group (67.4 ± 2.9 years of age) who maintained their daily lifestyles. All study participants were instructed to perform a selected pace walking for recording gait parameters (stride length, gait speed, swing cycle time, stance phase, and double support times) at both baseline and after the experiment. Low-limb flexibility and range of motion at specific musculoskeletal regions (hip flexion, hip extension, and plantar flexion, as well as anterior and lateral pelvic tilts, pelvic rotation, and joint range of motion (hip, knee, and ankle)) were also assessed in the present study. Multiple separate 2 × 2 Factorial Analysis of Variance (ANOVA) with repeated measures were used to examine the effects of TC on the abovementioned outcomes between baseline and posttest in the two groups. When compared to those in the control group, older female adults who experienced the 8-week Tai chi intervention demonstrated significant improvements in most of the outcome measures. More specifically, positive changes in the TC group were found, including gait parameter (p < 0.001 for all; stride length (1.12 to 1.24, +8.6%), gait speed (1.06 to 1.21, +13.9%), stance phase (66.3 to 61.8, −5.5%), swing phase (33.7 to 38.4, +10.1%), double support time (0.33 to 0.26, −21.1%)), flexibility-related outcomes (hip flexion (90.0 to 91.9, 22.6%, p < 0.0001), single hip flexor (6.0 to 2.0, −61.5%, p = 0.0386), and plantar flexion (41.6 to 49.7, +17.5%, p < 0.0001)), and range of motion (anterior pelvic tilt (9.5 to 6.2, −34.7%, p < 0.0001), lateral pelvic tilt (6.6 to 8.3, +23.8%, p = 0.0102), pelvic rotation (10.3 to 14.7, 28.2%, p < 0.0001), hip range of motion (29.8 to 32.9, +13.5%, p = 0.001), and ankle range of motion (28.0 to 32.6, +11.1%, p < 0.0001)). The present study supports the notion that the practice of TC has a positive effect on healthy older female adults in improving gait parameters and flexibility, counteracting the normal functional degeneration due to age.