Sepsis is a severe medical condition distinguished by immune systematic dysfunction and multiple organic injury, or even failure, resulting from an acute systemic inflammatory response. Acute liver injury (ALI) could be considered as a notable inflammatory outcome of sepsis. Studies have demonstrated the essential roles played by long non-coding RNAs (lncRNAs) in mediating the processes of various diseases, including their ability to engage in interactions with microRNAs (miRNAs) as complexes of competing endogenous RNA (ceRNA) to modulate signaling pathways. In this study, a newly discovered lncRNA, named 220, was identified to function in regulating autophagy and apoptosis in Kupffer cells treated with lipopolysaccharide (LPS). This was achieved through sponging miR-5101 as a ceRNA complex, as identified via high-throughput sequencing. The expression of 220 was found to be significantly different in the hepatic tissues of endotoxemic mice that were treated with LPS for 8 h, ultimately modulating the ALI process. Our studies have collectively demonstrated that 220 is a novel regulator that acts on LPS-induced autophagy and apoptosis in Kupffer cells, thereby mediating the ALI process induced by LPS. Furthermore, the validation of our findings using clinical databases suggests that 220 could potentially serve as a molecular target of clinical, diagnostic, and therapeutic significance in septic liver injury.