Protected areas (PAs) are essential for ensuring the sustainability of biodiversity and protecting human lives. Efficient and sustainable protection require cooperation of all stakeholders and anticipate the coherence of conservation aims and human use. PAs can limit the human activities and land use alternatives significantly. Participatory planning and monitoring can help to achieve the protection goals and 3D GIS can serve as the base platform for planning. This study investigates the potential of 3D GIS with high-resolution datasets for accurate definition of PA boundaries and demonstrates a case study in Cesme Peninsula, Turkey. The PA boundaries have been examined with respect to the property ownership boundaries and buildings using the 3D city model of Cesme. A conceptual approach for the determination of PA boundaries is proposed. The results show that 3D GIS can support the ecosystem conservation efforts while observing the interests of all stakeholders at the same time.