Introduction: The position of handlebar in drop handlebar is lower than the stem, while higher than the stem in bullhorn handlebar. Flat handlebars are horizontal to the stem. People cycling with different types of handlebar on diverse roads lead to different riding position, active different muscles of upper limb and cause various sport injuries when riding for hours. The aim of this study is to investigate the effect of bicycle handlebars on muscle activation and vibration. Methods: Eighteen college students were recruited. The DELSYS surface electromyography system (1000 Hz) and built-in three axes accelerometers (150Hz) were used to record the muscle activity and vibration amplitude (resultant acceleration) of extensor, flexor, bicep and tricep. Subjects were assigned to ride on the asphalt road or stone brick road with three different kinds of handlebars. The data were recorded as subjects reached at a steady cadence of 50 rpm and kept for at least 20 m. Two-way ANOVA was applied for the comparison between groups, and the statistical significance set at α =.05. Results: The muscle activation in flexor carpal muscle was significantly lower as volunteers cycling with flat handlebar than those with drop or bullhorn handlebar on both asphalt road and stone brick road. On asphalt road, the muscle activation in triceps brachii was higher as cycling with bullhorn handlebar than those with flat or drop handlebars. In vibration studies, as riding on both the asphalt road and stone brick road, the lowest vibration of flexor carpal muscle was noticed in the group using flat or drop handlebar. Cycling with flat or drop handlebar caused a higher vibration in triceps brachii muscle than that with bullhorn handlebar. The vibration of biceps brachii muscle was lower in those people riding with flat handlebar than those with bullhorn or drop handlebar on asphalt road. The highest vibration in extensor carpi ulnaris muscle was recorded as volunteers riding on the stone brick road with flat handlebar. On stone brick road, the vibration of biceps brachii muscle was lower in flat handlebar than that of drop handlebar. Conclusions: For more comfortable ride on the bike routes, we suggest bicyclist biking with the flat handlebars to separate the vibration in arms and trunk and decrease the activation of the upper limb.