Background: Isoflurane, one of the most commonly used inhalational anesthetics, is usually used in surgery patients and often causes long-term learning and memory impairment. The aim of this study was to explore the role of microRNA-204-5p (miR-204-5p) in isoflurane-induced learning and memory impairment in rats. Methods: The Morris Water Maze (MWM) test was used to estimate the spatial learning and memory abilities of laboratory rats. Enzyme-linked immunosorbent assay (ELISA) was used to determine interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) concentrations in the hippocampal tissues. The expression level of miR-204-5p was determined by using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The potential target genes of miR-204-5p were predicted and verified by the TargetScan and dual-luciferase reporter assay, respectively. Results: Isoflurane-induced rats showed significantly higher neurological function scores, higher escape latency and shorter time spent in the original quadrant. Isoflurane could significantly induce neuroinflammation, and the expression of miR-204-5p was increased in the hippocampal tissue of rats exposed to isoflurane. Moreover, downregulation of miR-204-5p attenuated the effect of isoflurane treatment on the escape latency and the time in the original quadrant, and inflammatory cytokines level was downregulated by inhibiting the expression of miR-204-5p. EphB2 was verified as a direct target gene of miR-204-5p. Conclusion: Downregulated miR-204-5p exerts protective effects against isoflurane-induced learning and memory impairment via targeting EphB2 and inhibiting neuroinflammation. MiR-204-5p could serve as a potential therapeutic target for the lightening of cognitive dysfunction induced by isoflurane.