Renal ischemia/reperfusion injury (RI/RI) is one of the major causes of acute kidney injury (AKI). Low molecular weight-dextran sulphate (LMW-DS) is a bioactive polysaccharide with anti-inflammatory and cytoprotective properties. This study investigated the nephroprotective potential of LMW-DS against the RI/RI. RI/RI rat model underwent surgery that involved a complete bilateral clamping of renal hila for 30 min, followed by unclamping for 30 min. LMW-DS (5 mg/kg, s.c.) was injected 5 min before unclamping. After 24 h, kidneys were collected for biochemical, histopathological, and immunohistochemical examinations. RI/RI dramatically induced renal deterioration, as reflected by marked elevation in the levels of renal oxidative stress, tumor necrosis factor-α, interleukin-6, and toll-like receptor-4, indicating lipid peroxidation and inflammatory response. Furthermore, RI/RI caused upregulation of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Additionally, morphological alterations and collagen deposition were also detected in kidney tissues. The immunohistochemical analysis revealed a strong positive immunoreaction for p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and caspase-3. Treatment with LMW-DS produced a pronounced amelioration in the altered parameters induced by RI/RI. In conclusion, LMW-DS exerts nephroprotective effects against RI/RI by restoring redox balance, suppressing inflammatory signaling, JNK/p38 MAPK, and caspase-3, highlighting its potential as a therapeutic strategy for renal I/R-induced AKI.