Roads and highways represent one of the most important anthropogenic impacts on natural areas and contribute to habitat fragmentation, because they are linear features that can inhibit animal movement, thereby causing barrier effects by subdividing the populations adjacent to the roads. The study presented here aims to determine, to which extent roads act as a barrier, subdividing populations of three species of small forest mammals: bank vole, yellow-necked mouse and common shrew, and what is the relative importance of road width and traffic intensity on the barrier effect. The study was carried out at four 25 m long segments of roads, close to the city of České Budějovice. All segments crossed a forest. The capture-recapture method was applied to determine the crossing rates of animals. The traps were checked three times each day during four consecutive nights, in summer and in autumn. We found that: (1) roads strongly prevent crossing movements in all three studied species, (2) there are interspecific differences in road crossing rates, (3) species cross more often narrow than wide roads, (4) traffic intensity does not affect the crossing rates.