Flip-flops may change walking gait pattern, increase muscle activity and joint loading, and predispose wearers to foot problems, despite that quantitative evidence is scarce. The purpose of this study was to examine the lower limb muscle co-contraction and joint contact force in flip-flops gait, and compare with those of barefoot and sports shoes walking. Ten healthy males were instructed to perform over-ground walking at self-selected speed under three footwear conditions: 1) barefoot, 2) sports shoes, and 3) thong-type flip-flops. Kinematic, kinetic and EMG data were collected and input to a musculoskeletal model to estimate muscle force and joint force. One-way repeated measures ANOVA was conducted to compare footwear conditions. It was hypothesized that flip-flops would induce muscle co-contraction and produce different gait kinematics and kinetics. Our results demonstrated that the musculoskeletal model estimation had a good temporal consistency with the measured EMG. Flip-flops produced significantly lower walking speed, higher ankle and subtalar joint range of motion, and higher shear ankle joint contact force than sports shoes (p < 0.05). There were no significant differences between flip-flops and barefoot conditions in terms of muscle co-contraction index, joint kinematics, and joint loading of the knee and ankle complex (p > 0.05). The variance in walking speed and footwear design may be the two major factors that resulted in the comparable joint biomechanics in flip-flops and barefoot walking. From this point of view, whether flip-flops gait is potentially harmful to foot health remains unclear. Given that shod walking is more common than barefoot walking on a daily basis, sports shoes with close-toe design may be a better footwear option than flip-flops for injury prevention due to its constraint on joint motion and loading.