Understanding human perceptions of public transport (PT) travel is essential for improving PT provision/operation and the travel experiences of PT users, as well as for encouraging more people to use green and sustainable travel to reduce the congestion, air pollution, and energy costs that many urban systems are facing. Based on behavioral experiments and surveys, existing research has revealed that the spatial and temporal characteristics of PT travel greatly influence human perceptions; however, neuroimaging evidence confirming these findings remains lacking. In this study, we conducted a functional magnetic resonance imaging- (fMRI-) based PT travel and recall experiment with 9 participants and collected 651 unique bus travel trajectories in Beijing City over 21 days. We extracted 22 spatial and temporal characteristics and derived brain activity patterns for each PT travel route. A representational similarity analysis (RSA) was performed to quantify the relationship between these spatial and temporal characteristics and brain activity patterns. Verbal descriptions were collected to cross-check the brain imaging results. We show that the participants' brain activity patterns were significantly correlated with seven spatial (i.e., circuity, turn, angular deviation, slope, point of interest (POI), and land use) and two temporal (i.e., travel time and waiting time) features, although individuals' perceptions of PT travel differed substantially. These findings are in line with the participants' verbal reports. Our findings offer new neuroimaging evidence from an interdisciplinary perspective on the perception of the spatial and temporal characteristics of PT travel, which not only lays an empirical basis but also provides a “neurourbanism” approach to improve travelers' subjective well-being during PT travel.