Objective To investigate the protective effect of isoflurane on renal ischemia reperfusion injury (IRI) and the related molecular mechanism. Methods According to random number table method, thirty‑two rats were divided into the sham group (S group), renal ischemia reperfusion injury group (IRI group), isoflurane preintervention group (Iso+IRI group) and isoflurane+lipopolysaccharide group (Iso+IRI+LPS group), with 8 rats in each group. The Iso+IRI group was pretreated with isoflurane (Iso), and the Iso+IRI+LPS group was intraperitoneal injected with lipopolysaccharide (LPS) 0.1 mg/kg after Iso pretreatment, the other groups were given the same volume of normal saline. Then renal IRI model was established by clamping renal pedicle method in IRI group, Iso+IRI group and Iso+IRI+LPS group, and the S group were treated with sham operation. The levels of creatinine (Cr), urea nitrogen (BUN) in venous blood were detected 24 h after operation, the levels of interleukin (IL)‑1β, tumor necrosis factor (TNF)‑α, IL‑6, in renal tissues were detected by enzyme linked immunosorbent assay (ELISA), the superoxide dismutase (SOD) activity in renal tissues were detected by SOD kit, the malondialdehyde (MDA) and glutathion peroxidase (GSH‑Px) were detected by visible spectrophotometry. The histopathological changes of kidney and pathological score were observed by hematoxylin‑eosin (H‑E) staining, while the apoptotic rate of renal tubular cells was detected by terminal deoxynucleotidyl transferase‑mediated dUTP‑biotin nick end labeling (TUNEL) method. The levels of Toll‑like receptor 4 (TLR4), nuclear factor (NF)‑κB p65 and myeloid differentiation factor88 (MyD88) were detected by Western blot. Results Compared with the S group: the levels of Cr, BUN, IL‑1β, TNF‑α, IL‑6, MDA, TLR4, MyD88, NF‑κB p65 and apoptosis rate in IRI, Iso+IRI and Iso+IRI+LPS groups were increased (P<0.05); the SOD activity in IRI group, Iso+IRI group, Iso+IRI+LPS group, and the levels of GSH‑Px in IRI group, Iso+IRI+LPS group were decreased (P<0.05). Compared with IRI group: the levels of Cr, IL‑1β, TNF‑α, IL‑6, MDA, TLR4, MyD88, NF‑κB p65 and apoptosis rate in Iso+IRI and Iso+IRI+LPS groups, and the pathological score and the levels of BUN in Iso+IRI group were all decreased (P<0.05); while the SOD activity and the levels of GSH‑Px in Iso+IRI group were increased (P<0.05). Compared with Iso+IRI group: the pathological score, the levels of Cr, BUN, IL‑1β, TNF‑α, IL‑6, MDA, TLR4, MyD88, NF‑κB p65, apoptosis rate were increased (P<0.05); the SOD activity and the levels of GSH‑Px were decreased in Iso+IRI+LPS group (P<0.05). Conclusion Iso ameliorates renal IRI by inhibiting TLR4/NF‑κB signaling.