User perception strongly influences transportation mode choice. Two case study cities, Kharagpur and Asansol, India, with different urban characteristics and bicycle patronage, were selected for analysis to capture the effects of urban characteristics such as city size, structure, economy, and demographic patterns on users' perception of bicycle mode choice. Eighteen factors influencing the choice of bicycling, either as motivators or as deterrents, were identified. Selected attributes were analyzed on the basis of user perception across different population subgroups to investigate their effect on the choice of bicycling as a travel mode. The Kruskal–Wallis H-test and the Mann–Whitney U-test were used to test for heterogeneity in user response across various population subgroups. The results of these tests indicated that perceptions of almost all attributes differed significantly across the two cities in this study. Hence, two models were developed with the use of exploratory factor analysis (EFA) to extract the underlying structure of user perception. Two five-factor models were derived from the analyses, with one factor, perceived benefits, being common to both cities, reflecting a similarity in users' attitudes toward benefits associated with bicycling. On the basis of the EFA results and user ranking, most significant variables were identified. Among them, physical fitness was identified as a motivator common to both cities. Route visibility, road width, and on-street parking were identified as deterrents common to both cities, indicating common concerns about road infrastructure among users. These key attributes could be used to formulate bicycle-related policies and could be included in stated-choice experiments for the purpose of valuation.