Abstract: To investigate the protective effect of sporoderm-removed Ganoderma lucidum spores (RGLS) against 5-fluorouracil (5-FU)-induced liver injury and to elucidate the underlying mechanisms. A 5-FU-induced liver injury model was established in C57BL/6 mice by intraperitoneal injection of 5-FU. The effects of RGLS on body weight, liver index, liver histopathology, serum markers of liver injury, oxidative stress indices, and expression of lipid-metabolism-related genes were evaluated. An in vitro liver injury model was generated by treating THLE-2 cells with 5-FU, and the effects of RGLS on cell viability, apoptosis, and the expression of apoptosis- and oxidative stress-related proteins were assessed. High-dose RGLS (RGLS-H, 1.6 g/kg) significantly attenuated 5-FU-induced liver injury, as evidenced by reductions in liver index, serum alanine aminotransferase (ALT) levels, and hepatic malondialdehyde (MDA) content, along with an increase in hepatic superoxide dismutase (SOD) activity (P<0.05). Histopathological analysis showed that RGLS-H mitigated 5-FU-induced hepatic steatosis and inflammatory cell infiltration. In addition, RGLS-H significantly down-regulated the hepatic mRNA expression of fatty acid synthesis genes (Acly, Scd1) and up-regulated the key fatty acid β-oxidation gene Cpt1a (P<0.05). In THLE-2 cells, RGLS significantly improved cell viability and reduced 5-FU-induced apoptosis (P<0.05). Mechanistically, RGLS decreased the expression of the pro-apoptotic proteins Bax and cleaved caspase-3, while increasing the expression of the anti-apoptotic protein Bcl-xL and the antioxidant proteins Nrf2 and HO-1 (P<0.05). In conclusion, RGLS protects against 5-FU-induced liver injury by inhibiting hepatocyte apoptosis and oxidative stress, likely through activation of the Nrf2/HO-1 pathway, and by correcting 5-FU-induced disturbances in hepatic fatty acid metabolism. These findings support the potential of RGLS as a hepatoprotective adjuvant during 5-FU-based chemotherapy.