This paper studies and compares the impacts of Madrid-Seville High-Speed Rail (HSR) on population growth in the five cities served by this line—Madrid, Ciudad Real, Puertollano, Córdoba, and Seville. The analysis period ranges from 1990 to 2006. The comparative analysis finds that the impacts of HSR largely differ. The association between the opening of HSR and population growth in Ciudad Real is observable; but the relationship is not clear in other station inner buffer areas. To study the impacts of HSR on population growth in each station area, accessibility-based spatial autoregressive models are adopted. The estimates suggest that HSR contributes to Ciudad Real with a relevant population increase, whereas it seldom benefits Puertollano. As the two largest cities along the HSR line, the improvement of accessibility leads to population growth in central Madrid. On the contrary, in Seville, instead of moving towards the central area, with easier access to suburban areas and to other cities, people are likely to move out from the city center. In Cordoba, population growth is not directly influenced by the improved accessibility, but is significantly correlated to the new residential land development, as the urban renovation project affiliated to HSR.