Inhaled anesthetic agents may be neurotoxic to the developing brain of a neonatal rodent. Isoflurane is a commonly used volatile anesthetic agent for maintenance of general anesthesia in various types of surgery. Neonatal exposure to isoflurane has been implicated in long-term neurocognitive dysfunction in children. The mechanisms of isoflurane-induced neurotoxicity have not been fully elucidated. Disruption of gut microbiota is currently attracting considerable interest as a vital pathogeny of some neurologic disorders. In the rat model, it is unknown whether neonatal exposure to isoflurane impacts the gut microbiota composition of juvenile animals. In the present study, postnatal 7-day-old male rats were exposed to 1 minimum alveolar concentration isoflurane for 4 h. Non-anesthetized rats served as controls. The fecal microbiomes of rats were observed using 16S RNA sequencing technique on postnatal day 42. Results indicated that composition of gut microbiota of isoflurane-exposed rats was different from controls. Several bacteria taxa in isoflurane-exposed rats were different from those of controls at various taxonomic levels. In particular, the abundance of Firmicutes, Proteobacteria, Clostridia, Clostridiales, and Lachnospiraceae were significantly increased in exposed rats and the abundance of Bacteroidetes, Actinobacteria, Bacteroidia and Bacteroidaceae were significantly decreased compared to controls. These results may offer new insights into the pathogenesis of isoflurane-induced neurotoxicity.