Accessibility plays a major role in achieving sustainable transport, and therefore urban and regional sustainability. The urban public transport system promotes mobility and realizes a large part of urban movements. Moreover, improving accessibility in order to promote sustainable transport requires the application of new concepts and indicators as a powerful tool in the process of creating a balanced urban transport system. In this regard, one of the main goals of this research is to present an overview of the relevant accessibility indicators and assessment of accessibility in regional Cross-Border Cooperation (CBC) in order to transcendence challenges and obstacles for sustainable transportation in these regions along of Portuguese-Spanish border. This paper focuses on the accessibility of cross-border cooperation scenarios along the border regions of Alto Alentejo (Portugal) and Badajoz (Spain) where the Case Study Research Method (CSR) made it possible to recognize accessibility as a key factor in territorial success. Also, accessibility analysis can assess improvements as well as regional imbalances. In addition, this methodology can be used to identify missing links, which requires new investments enabling long-term sustainability.