Bicycle use increases in many cities around the world. In the Netherlands, cycling is one of the main transport modes in cities and bicycle use is still growing. This leads to crowded cycling infrastructure in cities with high cycling shares, including in the four largest Dutch cities. Since few studies have been done to the effects of high crowding levels on cyclists’ route preferences and perceived safety, the present study aims to examine this for Dutch urban cyclists. Moreover, the relationship between perceived safety and route preferences is established. To investigate this, a questionnaire, including a route choice experiment, is completed by 1,329 cyclists from the four largest Dutch cities. The effects of varying crowding levels on route preferences and perceived safety are analysed with Mixed Logit models. Logistic regression is used to investigate the consistency between route preferences and perceived safety. The results show that crowding negatively affects route preferences as well as perceived safety, and that the impact is stronger for older cyclists and women. Furthermore, high crowding levels have a negative impact on the preference for and perception of safety of cycling infrastructure. Moreover, it is shown that all investigated route attributes have a significant effect on perceived safety, implying a more direct relationship between perceived safety and route preferences. In addition, the results show that most cyclists prefer routes they also perceive as safe. Concludingly, crowding seems an important issue for cyclists in large Dutch cities. Moreover, the perception of safety is likely to increase with the implementation of cycling infrastructure suitable for large flows of cyclists, leading to a safer cycling network for all types of cyclists.