Background: Having a valid tool to assess attitudes toward vaccination and identify the concerns that drive vaccine refusal can facilitate population studies and help guide public health interventions. The objective of this study has been to adapt the Vaccination Attitudes Examination (VAX) scale in Colombian university students and to study its psychometric properties in a non-probabilistic sample of 1074 Colombian university students. Methods: A confirmatory factor analysis was used to study the factorial structure. A structural equation model was tested to study concurrent validity and to check whether the factors predicted having received the coronavirus vaccine. Gender-based measurement invariance was also studied for the best model. Results: The results support the structure of four related factors. The composite reliability index was good for all the factors, but the average variance extracted was not as good for the second factor. There was strong measurement invariance by gender, and two factors are good predictors of being vaccinated or not. Conclusions: The VAX has shown construct and concurrent validity and is a reliable tool for evaluating attitudes towards vaccines in university students in Colombia. It may help guide the implementation of actions for the National Vaccination Plan and institutional policies.