* While marine environments are three-dimensional (3D) in nature, current approaches and tools for planning and prioritising actions in the ocean are predominantly two dimensional. Here, we develop a novel 3D marine spatial conservation prioritisation approach, which explicitly accounts for the inherent vertical heterogeneity of the ocean. This enables both vertical and horizontal spatial prioritisation to be performed simultaneously. To our knowledge, this is the first endeavour to develop prioritisation of conservation actions in 3D. * We applied the 3D spatial conservation prioritisation approach to the Mediterranean Sea as a case study. We first subdivided the Mediterranean Sea into 3D planning units by assigning them a z coordinate (representing depth). We further partitioned these 3D planning units vertically into three depth layers; this allowed us to quantify biodiversity (1,011 species and 19 geomorphic features) and the cost of conservation actions at different depths. We adapted the prioritisation software Marxan to identify 3D networks of sites where biodiversity conservation targets are achieved for the minimum cost. * Using the 3D approach presented here, we identified networks of sites where conservation targets for all biodiversity features were achieved. Importantly, these networks included areas of the ocean where only particular depth layers along the water column were identified as priorities for conservation. The 3D approach also proved to be more cost-efficient than the traditional 2D approach. Spatial priorities within the networks of sites selected were considerably different when comparing the 2D and 3D approaches. * Prioritising in 3D allows conservation and marine spatial planners to target specific threats to specific conservation features, at specific depths in the ocean. This provides a platform to further integrate systematic conservation planning into the wider ongoing and future marine spatial planning and ocean zoning processes.