The Colorado River basin (CRB; 627,824 km2) is the primary water source for southwestern North America. The 283,384 km2 water-exporting Upper CRB (UCRB) from the Colorado Plateau and Rocky Mountains provides water and hydroelectric power to the 344,440 km2 Lower CRB (LCRB) in the Basin and Range province. Long-regarded as a snowmelt-fed river system, half of the river’s baseflow is derived from groundwater, primarily through springs, which are bio-culturally and economically significant but highly threatened. We synthesized information on the distribution, ecohydrology, biodiversity, status, and socio-economic impacts of 20,872 reported CRB springs in relation to perennial streams, human population growth, and climate change. CRB springs were patchily distributed, with highest density in geologically complex settings. Despite low quality mapping data, most springs-influenced habitats were small (median = 0.04 ha; minimum estimated total area = 8.4 km2, 0.001% of CRB land area). Median discharge was low (0.10 L/s, N = 1687), but ranged up to 1800 L/s. Springs water quality depended on aquifer hydrogeology, elevation and residence time. Arizona springs produced 0.6 km3/yr of water (6% of its water budget). Data on >330 spring-dependent taxa (SDT) of plants and animals revealed 2/3 of CRB fish and many endemic aquatic and riparian invertebrates exist. Endemic SDT plant species richness was low but within-springs species density was orders of magnitude greater than in adjacent uplands, with vegetation varying by spring type. A strong negative relationship exists between elevation and wetland plant diversity, likely a Pleistocene-Holocene transition phenomenon related to climate, pH, and conductance. Ecological impairment exceeded 70% in many landscapes, with stressors primarily related to groundwater depletion and pollution, livestock management, flow abstraction, and non-native species introduction. Ensuring the ecological integrity and sustainability of CRB groundwater supplies and springs will require improved policy along, more thorough inventory, and local ecosystem rehabilitation.