Acetobacter diazotrophicus is a nitrogen‐fixing endophytic bacterium, originally isolated from sugarcane. Its colonizing ability was evaluated in high and low N‐fertilized sugarcane plants by inoculating stem‐cuts with a β‐glucuronidase marked A. diazotrophicus strain. Bacterial quantification by the most probable number technique showed a severe decrease of A. diazotrophicus cells in plants fertilized with high levels of nitrogen. The inoculated strain was detected inside low N‐fertilized sugarcane plants by histochemical staining of β‐glucuronidase and scanning electron microscopy. A. diazotrophicus was found mainly inside cortical cells of stems and inside xylem vessels. No β‐glucuronidase activity was observed in non‐inoculated plants. High nitrogen fertilization of fields might be a threat to maintaining naturally occurring endophytic associations.