The present study was conducted with the aim of finding a suitable delivery method for introducing endophytic bacteria into new sugarcane stalks. Stem pieces of cultivars NiF8 and Ni15 were inoculated with Herbaspirillum sp. strain B501gfp1 by the vacuum infiltration method. The inoculated bacteria showed higher levels of nitrogenase activity in cultivar Ni15 than NiF8. The population density immediately after inoculation (10⁷ CFU g [fresh weight]^-1) showed that strain B501gfp1 successfully entered the stem tissues of Ni15, where its presence was confirmed by fluorescence microscopic observations. Colonization was observed in the intercellular spaces and between the cell layers. The population of inoculated bacteria remained stable at 5 days after inoculation. The inoculated stems also showed adequately strong acetylene reduction activity compared to uninoculated stems in the absence of indigenous nitrogen-fixing bacteria.