In the Netherlands the increase in car travel not only puts a strain on the environment but the increase in congestions also has a negative effect on the accessibility of urban regions. The promotion of alternative transportation modes is a way to deal with these problems. Cycling is considered a sustainable alternative to motorized traffic for short trips. Over the past years a new group of cyclists has emerged called the e-bikers, making use of a vastly growing number of e-bikes. While the research in route choice behaviour of cyclists has grown manifold over the past years, the rise of e-bike users as a specific group seems to have been overlooked. This study adds to the understanding of route choice decisions of e-bike users by developing a route choice model using GPS data. For comparison a route choice model for bike users is estimated. The GPS data of 742 self-selected individuals corresponding to 17626 trips in the Noord Brabant region in the Netherlands collected during March 2014 was used to estimate a Multinomial Logit (MNL), Path Size Logit (PSL), Mixed Logit (ML) and Mixed Logit with Path Size Logit combination (ML+PSL), for trips made by bike and by e-bike. For both transportation modes the ML+PSL best described the data. The estimate for distance is significant and positive for both transportation modes. This indicates that the probability of a route being chosen increases when distance increases. This contradicts the base assumption of route choice modelling, and that people prefer the shortest route. A comparison between the two transportation modes indicates that several different variables, such as ‘weekday’ and ‘peak hours’, do have an effect on route choice when respondent make e-bike trips but don’t affect the route choice decisions when travelling by bike. The other way around, daylight has an effect on the route choice decision of bike trips but not on e-bike trip. The results of this study indicate that while the same person can be both an e-bike and bike user, depending on the trip, their route choice behaviour differs over the transport modes.