Fine Particulate Matter (PM2.5) has become a major issue in cities around the world as it adversely affects human health and the environment. This study aims to develop a deeper understanding of the impact of bicycle lanes designs on cyclist exposure to air pollution in a developing-country city. PM2.5 concentrations were measured along a predefined route with different bicycle lane designs in the city of Medellín, Colombia. The measurement campaign was made between October and December 2020 during peak and off-peak hours on weekdays, where a total of 29 bicycle trips were carried out. To obtain accurate measurements, we used a laser-based particle monitoring system. The study's findings reveal that the bicycle route section without dedicated bicycle lanes had the highest PM2.5 exposure and inhaled dose per kilometer traveled. Furthermore, the mean PM2.5 exposure for cyclists during the morning peak hours was higher (33.8 μg/m3) compared to the evening peak (16.1 μg/m3) and off-peak hours (11.1 μg/m3). The inhaled PM2.5 dose was three times higher during morning peak hours than during off-peak hours and twice as high during evening peak hours. These results show that segregated cycling lanes on the sidewalk can considerably lower PM2.5 exposure and inhaled doses for cyclists when compared to other lane designs, highlighting the significance of infrastructure development in supporting sustainable transportation and public health.