Resumo. The micropropagation is an important biotechnological tool for obtaining and maintaining mother grapevine plants with high quality plant health. The objective was to evaluate the establishment and multiplication in vitro and ex vitro acclimatization of grapevine cultivate Bordô in different culture media without adding growth regulators. Grapevine nodal segments were cultured in five culture media formulations without adding growth regulators. It was evaluated the number of leaves and roots, length of roots and shoots, replication rate, relative chlorophyll index, percentage of regenerated and rooted plants, dry biomass of shoot, root and total plants grown in vitro and after acclimatization. In vitro propagation of grapevine Bordô through nodal segments provided high rates of regeneration and rooting. High survival rates were obtained in the acclimatization of Bordô. Considering all the variables, culture media Roubelakis and Zlenko showed the best growth rates and development for shoots and roots, and in vitro multiplication rate of Bordô grapevine cultivar. Keywords: In vitro propagation. Vegetative propagation. Vitis.