With the growing interest in cycle tourism in Mexico, it is necessary to understand the motives for traveling and cycling. The aim of this study was to develop and validate the bicycle tourism motivation instrument (Cycle-tourist Motivation Instrument (CtMI)) for Mexico. The instrument was applied to 322 cycle tourists who participated, directly or indirectly, in the 2021 Chichimeca route, a cycle tourism event. Atypical data were removed, Cronbach’s alpha of the CtMi was 0.920, the Kaiser–Meyer–Olkin (KMO) test gave 0.911 as a result, and Barlett’s sphericity test was equal to 0.000; in addition, factor analysis with varimax rotation was performed with factor loadings greater than 0.40, resulting in an instrument with validity and explanatory capacity for the phenomenon of cyclist motivation with 32 items divided into 7 dimensions: health, social, competence domain, exploration, stimulus-seeking, self-presentation and escape, with values equal to and/or higher than those reported in the literature. The CtMI can contribute to future research related to this topic, which will allow us to understand and determine the motivations of the cycle tourist in Mexico. Likewise, this study demonstrates the need for further research to validate the CtMI in other contexts, within Mexico and abroad.