The ecosystem services framework is receiving increasing attention in the fields of policy and research. The assessment of human attitudes and perceptions regarding ecosystem services has been proposed as a promising tool for addressing complex problems associated with environmental change, particularly in the context of cultural landscapes. Transhumance is not only a farming practice responsible for shaping cultural landscapes but also an adaptive strategy based on mobility that may represent a useful approach to overcoming the growing challenges posed by accelerated environmental change. A socio-cultural valuation of ecosystem services associated with the Conquense Drove Road, one of the major transhumant networks still in use in Mediterranean Spain, was conducted via the distribution of questionnaires to 416 local residents and visitors to capture their perceptions regarding the importance of 34 ecosystem services (10 provisioning, 12 regulating, and 12 cultural) for both social and personal well-being. Overall, the ecosystem services considered to be the most important for social well-being were fire prevention, air purification and livestock. Most of the ecosystem services in question were perceived as declining, with the exception of those associated with recreation, scientific knowledge and environmental education. This study revealed that perceptions regarding the value of ecosystem services differed among respondents, depending on their age, place of origin and gender. Several methodological issues, as well as the implications of socio-cultural valuation for policy making, are also discussed here.