Since 2001, four emission measurement campaigns have been conducted in multiple traffic tunnels in the megacity of Saõ Paulo, Brazil, an area with a fleet of more than 7 million vehicles running on fuels with high biofuel contents: gasoline + ethanol for lightduty vehicles (LDVs) and diesel + biodiesel for heavy-duty vehicles (HDVs). Emission factors for LDVs and HDVs were calculated using a carbon balance method, the pollutants considered including nitrogen oxides (NO x ), carbon monoxide (CO), and sulfur dioxide, as well as carbon dioxide and ethanol. From 2001 to 2018, fleet-average emission factors for LDVs and HDVs, respectively, were found to decrease by 4.9 and 5.1% per year for CO and by 5.5 and 4.2% per year for NO x . These reductions demonstrate that regulations for vehicle emissions adopted in Brazil in the last 30 years improved air quality in the megacity of Saõ Paulo significantly, albeit with a clear delay. These findings, especially those for CO, indicate that official emission inventories underestimate vehicle emissions. Here, we demonstrated that the adoption of emission factors calculated under real-world conditions can dramatically improve air quality modeling in the region.