The variation of abiotic factors, and the plant species response to it, influence the regeneration capacity and, consequently, the structure of the forest community. The aim of this study is to describe the structure of the regenerating stratum and investigate its relationship with environmental and spatial variables along an altitudinal gradient. This study was carried out in the Santa Marta valley, state of Espírito Santo, Brazil. Data on height and diameter at soil height of regenerating individuals and environmental variables were collected in seven altitudinal quotas; and spatial variables (Moran’s Eigenvector Maps) were created based on geographic coordinates. Phytosociological parameters were calculated by species and the floristic similarity between the altitudinal quota were determined, as well as the species that characterize each group. Redundancy Analysis (RDA) was performed, and generalized linear models were adjusted to verify the influence of environmental and spatial factors on regenerating vegetation. The specie Palicourea sessilis presented the highest Importance Value. The formation of two floristic groups was verified, with 11 species indicative of the higher quota group (1,420 to 1,550 m) and only one of the second group (1,112 to 1,391 m). The first two axes of the RDA explained 51.02% of the variation in the floristic composition. The natural regeneration demonstrated environmental preferences, being influenced by luminosity, abundance in adult components, contents of calcium and sodium, plant litter accumulation, altitude, and spatial structure of the environment. The altitude did not seem to influence a pattern in abundance or richness of regenerating species.