Introduction: Cycling is a non-polluting and healthy transportation mode for older adults. However, there is limited knowledge about the infrastructural changes necessary to stimulate cycling among older adults (≥65 years). This is particularly true for electric cycling (e-biking), an increasingly popular form of cycling. The current experiment with manipulated photographs examined the environmental preferences for transportation cycling among older adults. Additionally, it examined whether subgroups with different environmental preferences exist and whether these subgroups differ on socio-demographics, health characteristics, transport behaviour, e-bike use and cycling levels. Methods: A structured questionnaire and choice-based conjoint exercise was completed by 895 Flemish older adults. The conjoint exercise included 13 choice tasks each presenting two street situations, which were manipulated on nine environmental attributes. Hierarchical Bayes and latent class analyses were applied to obtain environmental preferences and identify subgroups. Results: In the total sample, type of cycle path was the most important environmental attribute (importance=40.0, 95% CI=39.0–41.0) determining older adults’ preference for transportation cycling. The second most important attribute was traffic density (16.7, 95% CI=15.9–17.4), followed by cycle path evenness (11.8, 95% CI=11.4–12.1) and distance (10.6, 95% CI=10.1–11.0). Six subgroups with different environmental preferences were identified. These subgroups could be characterized based on differences in cycling limitations, driving status, ebike use and cycling levels. Conclusions: The provision of well-separated cycle paths should be considered a priority in urban planning initiatives aiming to stimulate transportation cycling among older adults. Such initiatives should be evaluated to validate the current findings and optimize future initiatives.