The Uquía Formation crops out in the Quebrada de Humahuaca in Jujuy province, Eastern Cordillera, NW Argentina. This unit is composed of a sequence of fluviatile sediments and water-laid air-fall tuff beds; it is approximately 260m thick and unconformably overlain by Pleistocene conglomerates and Quaternary alluvium. The sediments have been folded into a syncline and broken by several faults that generally trend northwest–southeast. Following Castellanos stratigraphy, we characterize three units (Lower, Middle, and Upper) of the Uquía Formation. Biochronologically, the Lower Unit is assigned to the late Chapadmalalan, the Middle Unit (“Uquian fauna”) to the late Vorohuean and Sanandresian, and the Upper Unit to the Ensenadan. Biostratigraphic evidence provides a calibration of important biochronologic events in the Great American Biotic Interchange (GABI), namely, the first appearances of Erethizon, Hippidion, and proboscideans at 2.5Ma (late Pliocene) in South America. Geological and paleobiological evidence suggest that during the late Pliocene, the area could have been a wide intermountain valley at 1400–1700m elevation, with a more humid environment than that of the present day and some wet–dry seasonality that permitted the coexistence of forest and open areas. Uquian mammals also indicate that northwestern Argentina and the Pampean region have represented distinct biogeographical areas since at least the late Pliocene.