〔Abstract〕 Objective To investigate the relationship between circular RNA homeodomain interacting protein kinase 3(circHIPK3) and the activation of rat microglia(RM) cells. Methods In vitro, RM cells were cultured and randomized into normal and oxygen-glucose deprivation/reperfusion(OGD/R) groups, and the expression level of circHIPK3 in each group was detected by RT-qPCR. The circHIPK3 lentiviral vector with puromycin resistance was constructed, and the overexpression(OE) group and negative control(NC) group were set up. The optimal multiplicity of infection(MOI) for RM cells was determined based on fluorescence expression, and puromycin was used to screen RM cells stably expressing circHIPK3. The cells of OE and NC groups were treated with OGD/R, and the expression levels of ionized calcium binding adaptor molecule 1(Iba-1) and eukaryotic tumor necrosis factor receptor superfamily(CD40) were detected by Western blot. The circHIPK3 translational protein potential was analyzed by the circRNAdb database, while the potential binding microRNAs on circHIPK3 were predicted by circBank and Starbase databases. Results OGD/R down-regulated circHIPK3 in RM cells(P<0.000 1). The sequencing results were accurate and the lentiviral vector of circHIPK3 was constructed successfully. The optimal MOI of RM cells was 80, puromycin at a concentration of 2 μg/ml was used to screen RM cell lines stably expressing circHIPK3. RT-qPCR results showed that the expression level of circHIPK3 was significantly higher in the OE group compared with the NC group(P<0.01). Western blot results revealed that the expression levels of Iba-1 and CD40 in the OE group were markedly lower than those in the NC group(P<0.05). Protein translation analysis showed that circHIPK3 encoded a polypeptide of 404 amino acids with two internal ribosome entry sites(IRES) and an open reading frame(ORF). Database analysis uncovered that circHIPK3 could target eight specific miRNAs, namely hsa-miR-3529-5p, hsa-miR-379-5p, hsa-miR-506-3p, hsa-miR-33, hsa-miR-450b-5p, hsa-miR-551b-3p, hsa-miR-193, and hsa-miR-508-3p. Conclusion The overexpression of circHIPK3 effectively suppresses OGD/R-induced activation of RM cells. It has the potential to encode peptides and may act as a miRNA sponge. These findings provide a foundation for further study of circHIPK3 functions.