Acanthamoeba castellanii is a free-living amoeba found mainly in humid environments and Arcobacter butzleri is an emerging zoonotic pathogen, both can establish in vitro endosymbiotic relationships in the absence of bacterial replication. We analyzed the localization of A. butzleri within A. castellanii establishing their association with endoplasmic reticulum vesicles and mitochondria. Through confocal microscopy, we observed that during the early stages of endosymbiosis, there is not colocalization between amoebic vacuoles containing A. butzleri and mitochondria or ER vesicles of A. castellanii. Considering that energy production of this bacterium occurs via metabolism of amino acids or the tricarboxylic acid cycle, these results contribute to explain the absence of bacterial replication, since A. butzleri would not have access to the nutrients found in endoplasmic reticulum vesicles and mitochondria. In addition, we observe that A. butzleri induces significantly the actin polymerization of A. castellanii during the early stages of endosymbiosis.