Road-barrier effects can lead to population isolation, with consequent negative outcomes for individuals and populations. Small mammals have been identified as particularly vulnerable to barrier effects, yet few studies have focused on subterranean species. Given the burrowing habit of these species, we hypothesized that roads block their movement and therefore the gene flow between roadside populations. The tiny tuco-tuco (Ctenomys minutus), a small subterranean rodent that inhabits the coastal plains of southern Brazil, was used as a model species to test this hypothesis. We used 14 microsatellites to genotype 80 individuals from four colonies (n = 20 per colony). We compared the population differentiation (FST, G″ST and DEST) and population structuring (STRUCTURE and GENELAND, and discriminant analysis of principal components) of two colony pairs, one pair divided by a road, and the other with no road or other potential barrier between the colonies (control). The results indicated higher genetic differentiation and structuring between the roadside colonies than in the control sites, although less evident than initially predicted. We concluded that the road reduced but did not halt the gene flow of C. minutus. Nevertheless, in view of the rapid economic development of the region, measures to ensure long-term gene flow, i.e., installation or retrofitting of crossing structures, should be considered. This study complements previous analyses of road-barrier effects on small mammals, suggesting that subterranean species such as C. minutus can cope with these barriers, at least in conditions similar to our study area.