Resumo. The current study aimed to analyse the tree species natural regeneration floristic composition in a Mountain Seasonal Semideciduous Forest fragment in the domain of the Atlantic Forest, in southeastern Brazil (20°45¿S, 42°55¿W) in order to subsidize secondary natural forest management. Ten sites were selected based on their aspects, slopes and topographic positions. Natural regeneration indexes were estimated for each tree species per size classes to obtain the Total Natural Regeneration Index (TNR) for each species. It was sampled a total of 3,516 individuals of 140 tree species varying from 10 cm to 5 m height with diameter at the height of 1.3 m (DBH) smaller than 5 cm. The largest numbers of individuals were sampled in the families Rubiaceae, Fabaceae Mimosoideae, Monimiaceae, Fabaceae Papilionoideae, and the richest families were Fabaceae Caesalpinoideae, Fabaceae Papilionoideae, Euphorbiaceae, Lauraceae, Myrtaceae and Rubiaceae. Psychotria sessilis, Siparuna guianensis, Anadenanthera peregrina and Piptadenia gonoacantha presented the largest TNR. The comparison of the actual regeneration study with that of prior evaluations suggests a continuous substitution of species from initial to more advanced stages. These results indicate that the protection of the remaining forest fragments is important to maintain or improve the biodiversity in this region.