Whole-Body Vibration (WBV) has been extensively investigated as a widely used training tool. However, previous studies mostly applied the WBV on synchronous or side alternating vibration platform devices. The present study was aimed to investigate the electromyographic activity of trunk and lower-extremity muscles as one stands on a flat floor, and during horizontal whole-body vibration (WBV). This was a comparative cross-sectional study. Sixteen healthy adults participated in the study. The electromyographic activity of the trunk and lower extremity muscles was collected while the participants stood on either a flat floor or a WBV device moving horizontally. Electromyography (EMG) was used to record the activity of trunk and lower extremity muscles (erector spinae, rectus abdominalis, rectus femoris, biceps femoris, tibialis anterior, and gastrocnemius). The rectus femoris, tibialis anterior, and gastrocnemius muscles showed significantly higher muscle activation on the horizontal WBV device than on the flat floor (p<0.05). In particular, the electromyographic activity of Lt. rectus femoris (23.0 vs 14.3), Rt. rectus femoris (32.7 vs 15.1), Lt. tibiailis anterior (19.0 vs 9.8) Rt. tibiailis anterior (17.8 vs 7.9), and of the Lt. gastrocnemius (41.5 vs 15.7), Rt. gastrocnemius (32.7 vs 13.0) increased on the horizontal WBV device than on the flat floor. The muscle activity of the rectus femoris, tibialis anterior, and gastrocnemius muscles proved to be higher when the participants stood on the WBV device vibrating in a horizontal direction than on the flat floor. Further studies need to investigate the clinical applicability of horizontal WBV.