Background Urbanisation in Latin America (LA) is heterogeneous and could have varying implications for infant mortality (IM). Identifying city factors related to IM can help design policies that promote infant health in cities. Methods We quantified variability in infant mortality rates (IMR) across cities and examined associations between urban characteristics and IMR in a cross-sectional design. We estimated IMR for the period 2014-2016 using vital registration for 286 cities above 100 000 people in eight countries. Using national censuses, we calculated population size, growth and three socioeconomic scores reflecting living conditions, service provision and population educational attainment. We included mass transit availability of bus rapid transit and subway. Using Poisson multilevel regression, we estimated the per cent difference in IMR for a one SD (1SD) difference in city-level predictors. Results Of the 286 cities, 130 had <250 000 inhabitants and 5 had >5 million. Overall IMR was 11.2 deaths/1000 live births. 57% of the total IMR variability across cities was within countries. Higher population growth, better living conditions, better service provision and mass transit availability were associated with 6.0% (95% CI -8.3 to 3.7%), 14.1% (95% CI -18.6 to -9.2), 11.4% (95% CI -16.1 to -6.4) and 6.6% (95% CI -9.2 to -3.9) lower IMR, respectively. Greater population size was associated with higher IMR. No association was observed for population-level educational attainment in the overall sample. Conclusion Improving living conditions, service provision and public transportation in cities may have a positive impact on reducing IMR in LA cities.