The current study was conducted to evaluate the effects of unstable footwear on spine kinematics and trunk muscle activity during bipedal stance. Therefore, spinal alignment and concurrent angular velocity measures were assessed in 27 asymptomatic adults while standing with unstable Masai Barefoot Technology (MBT) shoes and standard footwear, respectively, employing a multisegmental three-dimensional trunk model. Electromyographic (EMG) analyses included recordings of the rectus abdominis, internal oblique, external oblique, upper thoracic paraspinals, lower thoracic erector spinae, and lumbar erector spinae. Kinematic and EMG variables were compared for the two shoe conditions via paired Student's t-tests (α = 0.05). Results showed that wearing unstable MBT shoes increased flexion at the mid-thoracic level (0.8°; P = 0.001) and led to greater mean velocities of angular displacement at the thoracolumbar (11.2%; P = 0.003) and at the lumbopelvic (10.8%; P = 0.02) regions, accompanied by more lumbar erector spinae activity (18.2%; P = 0.003). Accordingly, using MBT shoes may have potential implications in promoting spine tissue health, notably at the low back area, through expected improvements in terms of muscle conditioning and/or motor performance. However, additional investigations are warranted to further examine the framework of unstable footwear constructions within prevention and rehabilitation settings.