Sepsis-induced cardiac dysfunction (SICD) is a major contributor to mortality in sepsis. Kinesin family member 13B (KIF13B) has been identified as a critical protective factor for metabolic disorder and cardiovascular disease; however, the role of KIF13B in SICD remains unknown. After introducing lipopolysaccharide or cecal ligation and puncture surgery to wild-type ( WT ) and global Kif13b knockout ( Kif13b −/− ) mice combined with lipopolysaccharide-treated neonatal rat cardiomyocytes, we found that KIF13B expression levels were markedly down-regulated in septic hearts and cardiomyocytes. Kif13b deletion exacerbated SICD progress with reduced cardiac contractile function and resulted in increased mortality, accompanied by promoted lipid accumulation, fibrosis, and mitochondrial impairment. Mechanistically, the loss of KIF13B enhanced the lysosomal degradation of the lipid-droplet-associated protein perilipin 5 (PLIN5), thus disrupting the mitochondrial localization of PLIN5 and then impairing cardiac lipid homeostasis and proper mitochondrial function. Nevertheless, cardiac-directed AAV9- PLIN5 gene therapy sufficiently corrected cardiac dysfunction, inhibited lipid accumulation, and reduced oxidative stress in Kif13b −/− mice with SICD. In summary, these findings provide a new insight into the molecular mechanism underlying the pathogenesis of SICD, highlighting the KIF13B/PLIN5 axis as a potential therapeutic target for the treatment of SICD.