The Santa Cruz Formation (SCF) in Río Chalía (Austral Basin, Patagonia, Argentina) is a well-exposed fluvial succession with abundant and diverse fossil vertebrates accumulated during the Miocene Climatic Optimum (MCO). Using facies analysis, characterization of stratigraphic architecture, U–Pb geochronology and vertebrate palaeontology, we assess the timing and interplay of controlling factors on the sedimentation, including tectonics, global sea level, climate and sediment supply. Throughout the succession, there occurred a constant aggradation of the floodplain-dominated fluvial system. Seven zircon U–Pb ages constrain the time of accumulation between c. 18 and 15.2 Ma, under a relatively constant sedimentation rate of 150  ±  50 m myr–1. The large number of fossil vertebrates indicates a Santacrucian fauna, showing no recognizable changes through the section. The basin-scale, low-gradient anastomosed fluvial system of the SCF records a period of about 3 myr of relatively constant environmental conditions controlled by continuous basin subsidence and high sediment supply conditioned by explosive volcanism together with weathering of uplifting terrains in the Andes. In addition, the system was influenced by a temperate to warm and subhumid climate favoured by the MCO before the onset of the Andean rain shadow, together to high global sea levels.