Groundwater is the most ubiquitous source of liquid freshwater globally, yet its role in supporting diverse ecosystems is rarely acknowledged^1,2. However, the location and extent of groundwater-dependent ecosystems (GDEs) are unknown in many geographies, and protection measures are lacking^1,3. Here, we map GDEs at high-resolution (roughly 30 m) and find them present on more than one-third of global drylands analysed, including important global biodiversity hotspots⁴. GDEs are more extensive and contiguous in landscapes dominated by pastoralism with lower rates of groundwater depletion, suggesting that many GDEs are likely to have already been lost due to water and land use practices. Nevertheless, 53% of GDEs exist within regions showing declining groundwater trends, which highlights the urgent need to protect GDEs from the threat of groundwater depletion. However, we found that only 21% of GDEs exist on protected lands or in jurisdictions with sustainable groundwater management policies, invoking a call to action to protect these vital ecosystems. Furthermore, we examine the linkage of GDEs with cultural and socio-economic factors in the Greater Sahel region, where GDEs play an essential role in supporting biodiversity and rural livelihoods, to explore other means for protection of GDEs in politically unstable regions. Our GDE map provides critical information for prioritizing and developing policies and protection mechanisms across various local, regional or international scales to safeguard these important ecosystems and the societies dependent on them.