There is a growing global agenda for sustainable transport systems. The transport sector is the fastest growing contributor of CO2 emissions, with 8.1 million tonnes emitted by transport in London annually. E-bikes are thought to be environmentally superior to other motorised modes of transport, and can expand the role of bikes in cities by overcoming common barriers to traditional cycling such as time, distance and topography. However, e-bikes are still a niche market in London compared to elsewhere in Europe, and it is imperative that the reasons for this are understood to assess whether e-bikes can become a mainstream transport mode. Understanding this will have implications for transitions from carbon-intensive transport systems to sustainable ones. Framed by the multi-level perspective (MLP) within transition theory, an online survey and series of semi-structured interviews in the form of an innovative ‘e-ride-along’ were completed within a case study context. These methods sought to establish which factors affected travel mode choice, explore the attitudes and perceptions towards e-bikes of non e-bike users of different demographics, identify the main barriers to e-bike use in London, and understand whether attitudes towards e-bikes change before and after trying one for the first time. Results show that the main barriers to e-bike use in London are cost, safety, negative social stigma and lack of cycle infrastructure, and that these barriers are likely to be experienced differently depending on demographics. Results also show that habitual associations with unsustainable travel patterns could make the transition to sustainable transport modes such as e-bikes challenging. E-ride-along participants found the e-bike experience fun and enjoyable, with perceptions generally becoming more positive after riding one. In order for e-bikes to transition from a niche to a mainstream transport mode, a range of interventions is required by cross-regime actors in order to overcome barriers to e-bike use in London.