One of the concerns surrounding the commercial release of genetically modified (GM) crops is the escape of transgenes into agricultural or semi-natural habitats through vertical gene flow, as this may cause environmental or economic problems. There is also the concern that GM crops may affect pollinators and the pollination services they provide. Despite the growing commercial interest of GM tomato (Solanum lycopersicum), gene flow has been assessed only sparsely in tomato. To evaluate the likelihood of gene flow from GM tomato plants to sexually compatible plants, and to assess whether bumblebee activity is affected by GM tomato, three experiments were conducted under greenhouse conditions, using a Bt-tomato expressing the insecticidal Cry3Bb1 protein as model system: (a) artificial crosses between a GM tomato line, two wild tomato relatives (Solanum hirsutum and Solanum nigrum) and a non-GM tomato variety; (b) bumblebee-mediated crosses between GM and non-GM tomato plants and (c) visual observations of bumblebees' feeding behaviour. No hybrids were obtained between the GM tomato line and S. hirsutum and S. nigrum. In an experimental design where non-GM receptor plants outnumbered GM plants by approximately 3:1, the bumblebee-mediated cross-fertilisation rate between GM and non-GM tomato plants was measured at 4.3 ± 5.47%. No significant differences in feeding behaviour of bumblebees foraging on GM and non-GM tomato plants were observed. Therefore, we conclude that: (a) the probability of transgene introgression between the GM tomato line used in this study and its wild relatives S. hirsutum and S. nigrum is negligible; (b) bumblebee activity can mediate cross-fertilisation between GM and non-GM tomato and (3) the Cry3Bb1-expressing tomato line tested does not adversely affect the feeding behaviour of bumblebees.