We estimate the effect of urban spatial structure on income segregation in Brazilian cities between 2000 and 2010. Our results show that, first, local density conditions increase income segregation: the effect is higher in monocentric cities and smaller in polycentric ones. Second, the degree of monocentricity-polycentricity also affects segregation: while a higher concentration of jobs in and around the CBD decreases segregation in monocentric cities, a higher employment concentration in and around subcenters located far from the CBD decreases segregation in polycentric cities. Third, results are heterogeneous according to city size: local density does not increase segregation in small (monocentric) cities, it increases segregation in medium size cities, and it decreases segregation in large (polycentric) cities. Finally, results also differ between income groups: while local density conditions increase the segregation of the poor, a more polycentric configuration reduces the segregation of the rich.