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Aggregate segregation in asphalt mixture is a bothersome engineering issue during pavement construction. The practitioners have some measures to mitigate the segregation potential based on experiences which, however, can only reduce the risk to a certain extent. In this research, the authors aim to contribute to the discussion in a rational non-empirical way, by using novel experimental and numerical techniques. A case study is carried out to investigate the vibration-induced segregation in asphalt mixtures, corresponding to the circumstance arising during material transportation to the construction site. A novel experimental test is conducted for evaluating the segregation characteristics of asphalt mixtures under vertical vibration in laboratory conditions. A numerical investigation based on discrete element method is further performed to study the phenomenon from a micromechanical point of view. The obtained experimental and numerical results indicate that vibratory loading induces aggregate size segregation in asphalt mixtures, and the degree of segregation is influenced profoundly by the adhesive properties of bituminous binders and the aggregate gradation.