This study's goal was to evaluate the pilot exclusive lane for motorcycles (Motovía) in Cali, Colombia, from the perception of its users in 2012-2013. A cross-sectional study using roadside surveys plus a qualitative component of individual semi-structured interviews was carried out. Road users' perceptions were investigated before and after implementation of lane dividers (LD) and were compared with other roads in the city. Perceptions were compared using chi-square tests. In the study, 293 motorcyclists, 111 cyclists, and 115 automobile drivers were interviewed. Following the installation of the LD, the majority of motorcyclists and cyclists reported that LD made easier the driving maneuvers and decreased travel time, in contrast to perceptions of automobile drivers (p < 0.001). For most motorcyclists, the Motovia was considered as safe and effective, approving its continuity and replication. Half of automobile drivers and cyclists did not approve the installation of the Motovia due to travel time (drivers) and security issues (cyclists). Motovia is an option to enhance motorcyclists' safety on the road. It must, however, offer clear circulation alternatives for automobile drivers. According to users' perception, it is not safe for bicycles and other human-powered vehicles to share lanes with motorcycles. Further research about vulnerable road users' infrastructure is required.