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Objective: To examine the relationship between temporal asymmetry and complexity of muscle synergy during walking using rhythmic auditory cueing (RAC) and the factors related to changes in muscle synergy during walking with RAC in survivors of stroke. Design: Cross-sectional study. Setting: Wards at 2 medical corporation hospitals. Participants: Forty survivors of stroke (N=40; mean age, 70.4±10.3 years; time since stroke, 72.2±32.3 days) who could walk without physical assistance. Interventions: Not applicable. Main Outcome Measures: The participants were assessed in a random block design under 2 conditions: comfortable walking speed (CWS) and walking with RAC. Single-leg support time, kinematics, and electromyograms were measured. Factors related to the complexity of muscle synergy (variance accounted for by 1 synergy [VAF1]) between the walking conditions were examined using hierarchical multiple regression analysis. Results: In the RAC condition, lower limb flexion and knee flexion angles, single-leg support time on the paretic side, and the symmetry index of single-leg support time were increased compared with those in the CWS condition. VAF1 was decreased in the RAC condition (73.9±0.15) compared with that in the CWS condition (76.9±0.13, P=.002). Hierarchical multiple regression analysis revealed that the change in VAF1 was explained by change in single-leg support time (R2=0.43, P=.002). Conclusions: The RAC condition demonstrated a more complex representation of muscle synergy than the CWS condition; the change in single-leg support time on the paretic side related to the changes in muscle synergy more than changes in lower limb angle. These findings can help in the walking-training concept to improve muscle synergy deficits in survivors of stroke.