Previous studies have shown convincing evidence for positive relationships between transportation cycling in adults and macro-scale physical environmental factors. In contrast, relationships are less consistent for more changeable, micro-scale environmental factors. The majority of existing studies used observational study designs, which cannot determine causality. The present mixed-methods study used manipulated photographs to determine causal relationships between micro-scale environmental factors and the environment's invitingness for transportation cycling. Further, interactions among environmental factors and moderating effects of gender, age and educational level were investigated. For this study, panoramic photograph of a street was manipulated on eight environmental factors: traffic, speed bump, general upkeep, evenness of the cycle path, vegetation, separation of motorized traffic, separation with sidewalk and cycle path width. Sixty-six middle-aged adults participated in the study and sorted the manipulated panoramic photographs from least to most inviting to cycle for transportation. Participants also provided qualitative data on how they sorted the streets. Multilevel cross-classified modelling was used to analyse the relationships between the environmental manipulations and the invitingness-scores. The qualitative data were deductively categorized according to the environmental factors. All environmental factors, except for separation with sidewalk, proved to have a significant main effect on the invitingness-score for transportation cycling. Cycle path evenness appeared to have the strongest effect on the invitingness. This effect was even stronger in an environment with good compared to poorly overall upkeep. Another significant interaction effect showed that the invitingness decreased when both separations along the cycle path were present compared to only a separation with traffic. No moderating effects of the demographic factors on these relationships were found. Qualitative data confirmed the observed quantitative relationships and added depth and understanding. Current study shows that the use of manipulated photographs can be an effective way to examine environment-physical activity relationships. Our findings indicate that evenness of the cycle path may be a crucial environmental factor when aiming to increase a street's invitingness for transportation cycling among middle-aged adults. The findings of our exploratory study could be used to develop an environmental intervention to determine if our findings are applicable to real changes in cycling behavior.