This paper aims to evaluate the floristic and structure of natural regeneration in a forest fragment located in the south of Espírito Santo State, as well as to verify the interaction between vegetation and environmental variables. The study was developed in Private Reserve of Nature Patrimony (RPPN) Cafundó, with twelve sample units (1 x 20m), where three classes based on size - Class 1 (plants between 0.1 and 1m high), Class 2 (plants between 1.1 and 3m high) and Class 3 (plants higher than 3m and less than 5cm DBH). This RPPN has a total area of 517ha, which approximately 358ha were used for allocation of experimental units. Individuals were classified according to ecological group and dispersion syndrome. Floristic diversity, equability, density and frequency of species were also calculated. Analysis of natural regeneration, floristic similarity between natural regeneration and the upper layer as well the regenerating layer interaction with environmental variables was performed through Canonical Correspondence Analysis. We found 678 individuals distributed in 73 morphospecies/species, with Actinostemon klotzschii and Goniorrhachis marginata being the most abundant. Sorensen's similarity between natural regeneration and the adult tree layer was 29%. The most dominant ecological groups were late secondary, while zoochory was the predominant dispersion syndrome among existing species. Shannon diversity index was 3.13 and equability 0.72. Goniorrhachis marginata, Actinostemon klotzschii and Psychotria carthagenensis species had correlation with carbon (C), phosphorus (P), declivity and canopy cover. We concluded that the natural regeneration component within studied area has a high richness and density of individuals when compared to studies developed in similar phytophysiognomies. The studied environmental variables seem to have little influence on the distribution of natural regeneration species in the RPPN, once only carbon, phosphorus, declivity and canopy cover have explained the species distribution within fragment.