Intestinal diseases often stem from a compromised intestinal barrier. This barrier relies on a functional epithelium and proper turnover of intestinal cells, supported by mitochondrial health. Mitochondria and lysosomes play key roles in cellular balance. Our previous researches indicate that biogenic selenium nanoparticles (SeNPs) can alleviate intestinal epithelial barrier damage by enhancing mitochondria-lysosome crosstalk, though the detailed mechanism is unclear. This study aimed to investigate the role of mitochondria-lysosome crosstalk in the protective effect of SeNPs on intestinal barrier function in mice exposed to lipopolysaccharide (LPS). The results showed that LPS exposure increased intestinal permeability in mice, leding to structural and functional damage to mitochondrial and lysosomal. Oral administration of SeNPs significantly upregulated the expression levels of TBC1D15 and Fis1, downregulated the expression levels of Rab7, Caspase-3, Cathepsin B, and MCOLN2, effectively alleviated LPS-induced mitochondrial and lysosomal dysfunction and maintained the intestinal barrier integrity in mice. Furthermore, SeNPs notably inhibited mitophagy caused by adenovirus-associated virus (AAV)-mediated RNA interference the expression of TBC1D15 in the intestine of mice, maintained mitochondrial and lysosomal homeostasis, and effectively alleviated intestinal barrier damage. These results suggested that SeNPs can regulate mitochondria-lysosome crosstalk and inhibit its damage by regulating the TBC1D15/Fis1/Rab7- signaling pathway. thereby alleviating intestinal barrier damage. It lays a theoretical foundation for elucidating the mechanism of mitochondria-lysosome crosstalk in regulating intestinal barrier damage and repair, and provides new ideas and new ways to establish safe and efficient nutritional regulation strategies to prevent and treat intestinal diseases caused by inflammation.