This study aims to investigate the mechanism of baicalein (BAI) in treating sepsis-associated acute kidney injury (SA-AKI) based on network pharmacology and in vivo experimental validation. Network pharmacology was used to predict the core targets and signaling pathways of BAI in the treatment of SA-AKI, with molecular docking employed to validate the binding affinity of these targets. An SA-AKI mouse model was constructed, and in vivo experiments were conducted to evaluate the protective effects of BAI and the underlying mechanisms. The results identified 41 intersecting targets related to BAI, SA-AKI, and endoplasmic reticulum stress (ERS), with five core targets selected: tumor protein p53 (TP53), AKT serine/threonine kinase 1 (AKT1), B-cell lymphoma 2 (BCL-2), hypoxia-inducible factor 1α (HIF-1α), and cysteinyl aspartate specific proteinase-3 (Caspase-3). These targets were involved in apoptosis, HIF-1 and advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE) signaling pathways. Molecular docking results showed that BAI could spontaneously bind to these five core target proteins. In vivo experiments revealed that BAI reduced the levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), serum creatinine (Scr), blood urea nitrogen (BUN), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in SA-AKI mice. Moreover, BAI alleviated ERS and apoptosis in the kidneys of SA-AKI mice. Immunohistochemical analysis showed that BAI upregulated the expression of vascular endothelial cadherin (VE-cadherin) and downregulated intercellular adhesion molecule-1 (ICAM-1) expression. Western blot analysis demonstrated that BAI decreased the expression of HIF-1α, phosphorylated phosphoinositide 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), and phosphorylated mechanistic target of rapamycin (p-mTOR) proteins in SA-AKI mice. Overall, the results suggest that BAI may improve ERS, inhibit apoptosis, and reduce inflammation through the modulation of the PI3K/AKT/mTOR/HIF-1α signaling pathway, thereby protecting endothelial cell and mitigating SA-AKI.