Nobiletin (NOB), a plant-based polymethoxyflavone, is a promising protective agent against sepsis; yet the mechanisms were not fully elucidated. The gut microbiota is found to be strongly associated with sepsis-associated acute liver injury (SALI). Here, our study aimed to evaluate the protective effect of NOB on SALI and explore the underlying molecular mechanisms. Cecal ligation and puncture (CLP) was used to induce SALI in mice. NOB was administered by gavage for 7 days before CLP induction. The 16S rRNA gene sequencing and fecal microbiota transplantation (FMT) were performed to verify the function of the gut microbiota. The markers of ferroptosis, inflammation, gut microbiota composition, and liver injury were determined. NOB administration significantly alleviated hepatic ferroptosis and inflammation in septic mice. Meanwhile, NOB upregulated the expression levels of nuclear factor E2-related factor 2 (Nrf2) and its downstream protein heme oxygenase-1 (HO-1). The protective effect of NOB administration against ferroptosis in SALI mice was reversed by the Nrf2 inhibitor ML385. Additionally, increased abundances of Ligilactobacillus, Akkermansia, and Lactobacillus, and decreased abundances of Dubosiella and Bacteroides in the gut were observed under NOB administration, suggesting that NOB might modulate the gut microbiota composition of septic mice. Furthermore, gut microbiota ablation by antibiotic treatment partly reversed the protective effects of NOB on sepsis. FMT also confirmed that NOB inhibited ferroptosis and activated Nrf2 signalling in SALI mice by modulating the gut microbiota. These results revealed that, by modulating the gut microbiota, NOB attenuated ferroptosis in septic liver injury through upregulating Nrf2-Gpx4. Our findings provide novel insights into microbiome-based therapeutic approaches for sepsis.