The intestinal epithelial barrier plays a fundamental role in human and animal health. Mitochondrial dysfunction can lead to intestinal epithelial barrier damage. The interaction between mitochondria and lysosomes has been proved to regulate each other's dynamics. Our previous studies have demonstrated that biogenic selenium nanoparticles (SeNPs) can alleviate intestinal epithelial barrier injury through regulating mitochondrial autophagy. In this study, we hypothesize that the protective effects of SeNPs against intestinal epithelial barrier dysfunction are associated with mitochondrial-lysosomal crosstalk. The results showed that lipopolysaccharide (LPS) and TBC1D15 siRNA transfection both caused the increase of intestinal epithelial permeability, activation of mitophagy, and mitochondrial and lysosomal dysfunction in porcine jejunal epithelial cells (IPEC-J2). SeNP pretreatment significantly up-regulated the expression levels of TBC1D15 and Fis1, down-regulated Rab7, caspase-3, MCOLN2 and cathepsin B expression levels, reduced cytoplasmic Ca2+ concentration, effectively alleviated mitochondrial and lysosomal dysfunction, and maintained the integrity of the intestinal epithelial barrier in IPEC-J2 cells exposed to LPS. Furthermore, SeNPs obviously reduced cytoplasmic Ca2+ concentration and activated the TBC1D15/Fis/Rab7-mediated signaling pathway, shortened the contact time between mitochondria and lysosomes, inhibited mitophagy, maintained mitochondrial and lysosomal homeostasis, and effectively attenuated intestinal epithelial barrier injury in IPEC-J2 cells transfected with TBC1D15 siRNA. These results indicated that the protective effect of SeNPs on intestinal epithelial barrier injury is closely associated with the TBC1D15/Rab7-mediated mitochondria-lysosome crosstalk signaling pathway.