The topics of regional innovation systems (RIS) and cross-border regions attract increasing attention, but few studies combine the themes. Further, the existing empirical studies of cross-border innovation and knowledge creation analyse one case at one point in time, thus, making it difficult to assess the progress of integration in the regions, as well as the effect of cross-border innovation policies. Consequently, important questions are left unanswered, including the central research question of this paper: does the sudden removal of significant physical barriers directly impacts collaboration activity in cross-border innovation systems? This paper examines regional integration in the Oresund Region over time. It deals with a specific part of the RIS, as it analyses research collaboration between actors from the Danish and Swedish sides, with a specific emphasis on the biotech industry. Scientific collaboration constitutes an important element of a RIS, particularly in research-intensive sectors. The quantity and quality of co-authorships are used as indicators for research collaboration. The results indicate that the removal of internal barriers in a cross-border region can have a substantial and long-term positive effect on knowledge flows if a targeted policy effort is made. In the absence of such policies, no effect is found.