The development of alternative environmentally friendly modes of transportation is becoming an increasingly promising solution in traffic-congested and polluted urban areas. E-bikes, as one of them, are recognized as an ecologically sustainable means of transportation that has significant potential to replace motorized modes of transportation that can improve urban mobility. Relying on artificial intelligence and considering an ecological approach when considering the acceptability of e-bikes by setting a direct question for users influences the development of an innovative way of understanding and evaluating the use of more sustainable modes of transportation. In this regard, this study aims to elucidate the main variables influencing the acceptability of e-bike use using artificial neural network (ANN) models—multilayer perceptron (MLP) and radial basis function (RBF). For training and testing the models, data from a random sample obtained through an online questionnaire, which was answered by 626 residents of Belgrade (Serbia), were used. A multilayer perceptron with nine and seven neurons in two hidden layers, respectively, hyperbolic tangent activation function in the hidden layer and identity function in the output layer, gave better results than the radial basis function model. With an accuracy of 89%, a precision of 83%, a recall of 79%, and an area under the receiver operating characteristic (ROC) curve of 0.927, the multilayer perceptron model recognized the influential variables in predicting acceptability. The results of the model indicate that the mileage traveled, the frequency of motorcycle use, the respondents’ awareness of the pollution in Belgrade, and the age of the respondents have the greatest influence on the acceptability of using e-bikes. In addition to majority acceptability (69.8%), the results obtained by the model can represent a useful basis for decision-makers when defining strategies for the development and application of e-bikes while reducing traffic congestion and environmental pollution in Belgrade.