AIM: To explore the role of Pioglitazone (Pio) on a mouse model of retinal ischemia/reperfusion (I/R) injury and to elucidate the potential mechanism. METHODS: Retinal ischemia was induced in mice by increasing the intraocular pressure, and Pio was administered 4h though periocular injection before I/R. The number of cells in the ganglion cell layer (GCL) was counted 7d after retinal I/R injury. Glial fibrillary acidic protein (GFAP), nuclear factor-kappa B (NF-κB), p38, phosphorylated-p38, PPAR-γ, interleukin-1β (IL-1β), Toll-like receptor 4 (TLR4), NLRP3, cleaved caspase-1, caspase-1 were determined by real-time polymerase chain reaction and Western blotting. RESULTS: Pio promoted the survival of retinal cells in GCL following retinal I/R injury (P<0.05). Besides, retinal I/R injury stimulated the expression of GFAP and TLR4, which were partially reversed by Pio treatment (P<0.05). Retinal I/R injury-upregulated expression of NLRP3, cleaved caspase-1, IL-1β was attenuated after Pio treatment (P<0.05). Moreover, I/R injury induced activation of NF-κB and p38 were inhibited by Pio treatment (P<0.05). CONCLUSION: Pio promotes retinal ganglion cells survival by suppressing I/R-induced activation of TLR4/NLRP3 inflammasomes via inhibiting NF-κB and p38 phosphorylation.