Roadkill estimates for different species and species groups are available for many countries and regions. However, there is a lack of information from tropical countries, including from Latin America. In this study, we analyzed medium and large-sized mammal roadkill data from 18 toll road companies (TRC) in São Paulo State (6,580 km of monitored toll roads), Brazil. We extrapolated these roadkill numbers to the entire system of major paved roads in the State (36,503 km). The TRC collected mammal-road- mortality data both before (2-lanes) and after (4-lanes) road reconstruction. We used the “before” data from the TRC to estimate annual mammal-road-mortality along 2-lane roads that remained public roads. Combined with the data for the new 4-lane highways, this allowed us to estimate annual mammal road mortality for all the paved roads in the State. During 10 years of roadkill monitoring along toll roads, a total of 37,744 roadkilled mammals were recorded, representing a total of 32 medium to large-sized mammal species (average number of roadkilled individuals/year = 3,774 ± 1,159; min = 1,932; max = 5,369; 0.6 individuals roadkilled/km/year). Most roadkilled species were common generalists, but there were also relatively high roadkill numbers of threatened and endangered species (4.3% of the data), which is a serious conservation concern. Most of the roadkill was reported occurred during the nocturnal period (66%, n = 14,189) and in the rainy months (October–March) (55%, n = 15,318). Reported mammal roadkill tended to increase between 2009 and 2014 (R2 = 0.614; p = 0.065), with an average increase of 313.5 individuals/year. Extrapolation of the results to the entire São Paulo State, resulted in an average estimate of 39,605 medium and large-sized mammals roadkilled per year. Our estimates of the number of roadkilled individuals can be used as one of the input parameters in population viability analyses to understand the extinction or extirpation risk, especially for threatened and endangered species.