The Achilles tendon (AT) plays a crucial role in locomotion. Differences in AT morphological and mechanical properties between men and women may therefore contribute to sex-differences in tendon behavior. This study aimed to investigate sex-based differences in AT strain behavior and derived mechanical properties during walking. Seventeen men and fifteen women walked shod at their preferred velocity on an instrumented treadmill. Gait kinematics and AT displacement were captured synchronously using 3D motion capture and ultrasound imaging, respectively. A MANOVA was used to examine sex differences in morphological and mechanical properties (AT force, stress, strain, stiffness, elastic modulus, hysteresis, strain energy) and a separate one-way ANOVA used to examine walking parameters. Time-series data (AT force, displacement, strain, AT loading rate, shortening velocity, instantaneous AT moment arm length and ankle angle) were assessed with 1D statistical parametric mapping. Results indicated that men had significantly greater AT dimensions and stiffness than women. Women exhibited greater peak AT displacement and strain during the push-off phase, while AT loading rates were higher in men in late stance. These findings suggest potential sex-specific differences in AT behavior during dynamic activities, providing further insights into tendon biomechanics. Future research should explore AT mechanics under higher loading conditions and across different age groups to comprehensively understand sex differences in tendon behaviour.