Listeriosis is a foodborne disease caused by the Gram-positive bacterium Listeria monocytogenes, a pathogen that modulates its intracellular survival via vacuolar escape and cytosolic replication. In the present study, we examined the ability of 58 L. monocytogenes isolates recovered in Brazil (beef, clinical and environmental samples, from 1978 to 2013) to invade, replicate and spread in a human intestinal epithelial cell line (Caco-2). Premature stop codons were common in the inlA gene of serotype 1/2c strains from beef and environment samples, associated with decreased Caco-2 cell invasion when compared to other serotypes. The isolates varied widely in their intracellular doubling times, and there was no clear relationship between serotypes and samples origin. Serotype 1/2a isolates were generally impaired in their ability to spread between Caco-2 cells, with an average 30 % smaller focus area than the 10403S reference strain. However, most isolates of serotype 1/2b exhibited enhanced cell-to-cell spread, with an average 35 % increase in focus area. Our findings are consistent with serotype being a better predictors of cell invasion potential and cell spread compared with sample origin of isolates, although the most invasive isolates were primarily isolated from beef. Additionally, we have identified isolates that could provide novel insight into the pathogenicity of L. monocytogenes that may not be revealed by studying common laboratory reference strains.