Objective To investigate the protective effects and molecular mechanisms of thymosin β4 (Tβ4) on pyroptosis in BV2 microglial cells. Methods BV2 cells were divided into three groups: control group (no treatment), pyroptosis group [stimulated with 1 μg/mL lipopolysaccharide (LPS) for 12 hours, followed by 10 μmol/L nigericin (Nig) treatment for 1 hour], and Tβ4 treatment group (co-incubated with LPS and Nig, then treated with 1 μg/mL Tβ4 for 1 hour). An in vitro sepsis-associated encephalopathy model was established by LPS and Nig co-treatment. Viability of BV2 cells was assessed by CCK-8 assay. RT-qPCR was performed to detect mRNA expression of interleukin (IL)-1β, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), and interferon-β (IFN-β). IL-1β levels in cell supernatants were measured by ELISA. Protein expression of NLRP3, GSDMD-N, cleaved caspase-1, phosphorylated stimulator of interferon genes (p-STING), and phosphorylated interferon regulatory factor 3 (p-IRF3) was analyzed by Western blot. Cell death rate and mitochondrial reactive oxygen species (ROS) levels were detected by flow cytometry using propidium iodide staining and MitoSOX indicator, respectively. Results Compared with the pyroptosis group, Tβ4 treatment alleviated morphological damage caused by pyroptosis in BV2 cells. Intracellular mRNA expression of IL-1β, IFIT1, and IFN-β; IL-1β concentration in supernatant; protein expression of NLRP3, GSDMD-N, cleaved caspase-1, p-STING, and p-IRF3; cell death rate; and mitochondrial ROS levels were significantly decreased (P0.05). Conclusions Tβ4 protects BV2 microglial cells against LPS and Nig-induced pyroptosis by inhibiting oxidative stress and inflammatory responses, potentially through regulation of the cGAS-STING signaling pathway.