Virtual reality (VR) offers new opportunities to promote active behaviors by enhancing engagement and allowing controlled modifications of urban environments. This study investigates whether virtual environments (VEs) can evoke affective responses comparable with real environments (REs), both psychologically and physiologically, by using an immersive VE combined with a walking simulator that replicates walking motion. Forty-nine healthy adults, Luxembourg residents or cross-border commuters, aged 18–65, including students, university staff, and the general public, walked two contrasting street segments, walking-friendly and car-friendly, in both RE and VE in a crossover design. Affective responses were assessed through questions on aesthetics, safety, enjoyment, comfort, relaxation, momentary stress, and real-time physiological data collected using E4 wristband. Significant differences emerged between the RE and VE across all affective measurements, except for nonspecific skin conductance responses, with the RE consistently eliciting more positive affective responses. Nevertheless, similar affective trends were observed in both the RE and VE across the two segments. Moreover, environmental characteristics significantly influenced affective responses in both the RE and VE, with the walking-friendly segment yielding more positive affective ratings than the car-friendly one. The interactions between environment type (RE vs. VE) and segment type (car-friendly vs. walking-friendly) were not significant for most measurements, indicating that the effect of environment type on affective responses remained consistent across segments. These findings emphasize that VEs can mimic the overall patterns of affective responses observed in REs. This research highlights VR’s potential in planning healthier cities, offering insights into its benefits and limitations for future research. • VR walks showed lower affective ratings than real-world walks. • VR replicated the overall affective trends observed in the real-world environment. • Car-friendly street segment triggered higher arousal in the VR and real environment. • VR is a valid tool for assessing human-environment interactions.