Metabolic profiling via gas chromatography coupled to mass spectrometry was used to investigate the influence of endophytic bacteria on shoots of in vitro-grown poplar plants free from culturable endophytic bacteria. The results demonstrate that the occurrence of an endophytic Paenibacillus strain strongly affects the composition of the plant metabolites of in vitro-grown poplars. Eleven metabolites were significantly changed between inoculated and non-inoculated poplar plants as determined by two independent experiments. Detected shifts in the primary metabolism of the poplar plants pointed to a mutualistic interaction between bacteria able to fix nitrogen and the host plant with altered nitrogen assimilation patterns. The corresponding metabolic signature comprises increased asparagine and urea levels as well as depleted sugars and organic acids of the tricarboxylic acid cycle. These observations coincide with the fact that the Paenibacillus sp. strain P22 is able to grow without nitrogen in the medium, indicating nitrogen fixation from the air also known from other Paenibacillus spp. In combination with the detected plant-growth-promoting effects of the endophyte Paenibacillus P22, a novel mutualistic interaction is observed.