An updated conceptual model of groundwater flow of carbonate fractured aquifers of Apennine basin of the Chienti River (Marche Region) has been developed. This model derives from the application of classical hydrogeological approach, which is based on a detailed field survey, by upscaling methodology, aimed to localize the main spring locations (15 springs) and to evaluate their mean discharge (total discharge of about 3280 L/s). Acquired data have been combined with the hydrogeological evaluation of the geological-structural setting, for defining the hydrogeological role of the lithological complexes and of the structural setting (faults and thrusts), conditioning groundwater flow direction and amount. The geological-structural setting is typical of Central Apennine in the Umbria-Marchean succession. The bottom carbonate unit corresponds to the carbonate platform, overlapped by calcareous complexes having marly beds and marly-clayed complexes of pelagic facies, by a total thickness of about 2500–3000 m. Compressive tectonic processes, in a E-verging thrust-and-fold belt, combined with intense translational movements gave rise to significant overthrusts at regional scale. The cores of anticlines host large basal aquifers, hydraulically separated by low-permeability boundary due to marly and clay complexes, respect with the surrounding Scaglia aquifers. Groundwater flow moves mainly along N-S direction, parallel to the main thrusts, which act as no-flow limit. The proposed hydrogeological model includes seven local aquifers. There are two independent basal aquifers, feeding a deep groundwater flow in the basal carbonate complexes, including areas out of the hydrographic basin of Chienti River. Spring fed by these basal aquifers have a mean discharge of 1460 L/s. Additional five aquifers characterized by less deep and more local groundwater flow correspond to the Scaglia complexes, with a mean discharge of about 1820 L/s. The extension and the limits of all these aquifers have been verified by the evaluation of effective infiltration values, to define the recharge area of each spring group. The resulting conceptual model has been independently validated by chemical and isotopic analyses of groundwater, which confirmed or require modification of the preliminary model obtained by the hydrostructural approach.