Abstract: Objective To investigate the role of μ-opioid receptor (MOR) in Herkinorin-mediated regulation of oxygen-glucose deprivation/reoxygenation (OGD/R) injury in primary rat neurons and its relationship with β-arrestin2. Methods Primary rat neurons were divided into a control group, an OGD/R group, and Herkinorin-treated groups (0.1, 0.5, and 1 μmol/L). An OGD/R model was established by 24 h pre-incubation, 2 h oxygen–glucose deprivation, and 24 h reoxygenation. Herkinorin was added at the beginning of pre-incubation and immediately at the onset of OGD/R to achieve the designated final concentrations. To further explore the relationship between MOR and β-arrestin2, β-arrestin2 was knocked down using lentiviral transfection. Neuronal apoptosis was assessed by flow cytometry, and the levels of IL-6 and TNF-α in the culture supernatant were measured by ELISA. Western blot was used to detect the expression of MOR and κ-opioid receptor (KOR) in the cell membrane and cytoplasm, while immunofluorescence was employed to observe their intracellular distribution.The effects of Herkinorin on MOR distribution and OGD/R-induced injury after β-arrestin2 knckdooun were evaluated. Results Compared with the control group, the OGD/R group exhibited significantly increased neuronal apoptosis and elevated secretion of IL-6 and TNF-α, with a significant decrease in cytoplasmic MOR expression (P<0.05). Herkinorin significantly reduced neuronal apoptosis and inflammatory cytokine release in OGD/R neurons in a concentration-dependent manner (P<0.05). Neurons treated with Herkinorin (1 μmol/L) showed decreased MOR levels on the cell membrane and increased MOR levels in the cytoplasm (P<0.05).No significant differences were observed in KOR expression in either the cell membrane or cytoplasm among the groups (P>0.05). After β-arrestin2 knockdown, the shRNA-β-arrestin2 group showed an elevated apoptosis rate, increased membrane MOR expression, and decreased cytoplasmic MOR expression compared with the shRNA-NC group (P<0.05). Conclusions Herkinorin may alleviate OGD/R-induced injury in primary rat neurons by regulating the distribution of MOR between the cell membrane and cytoplasm, which may be closely associated with β-arrestin2.