Abstract: To investigate the protective mechanism of bacillus licheniformis (BL) exposed to a 13.5 d spaceflight aboard Shijian-19 (SJ19) satellite against lipopolysaccharide (LPS)-induced intestinal barrier damage in Caco-2 cells, transepithelial electrical resistance, fluorescein sodium flux, inflammatory factors and oxidative stress markers levels, tight junction and adherent junction proteins expression and key proteins expression in the TLR4-NF-κB signaling pathway were measured. The results indicated that BL restored the LPS-induced intestinal barrier function of Caco-2 cells, as evidenced by a 26.20% increase in TEER, a 1.09% decrease in fluorescein sodium flux, a reduction in the levels of TNF-α, IL-6, and IL-1β by 17.36%, 30.52%, and 20.89% respectively, a decrease in H₂O₂ and MDA levels of 12.16% and 35.98% respectively, and an increase in SOD activity of 6.21%. Furthermore, it significantly upregulated the junction proteins (Occludin, Claudin-1, ZO-1, β-catenin, and E-cadherin) expression and suppressed the TLR4-NF-κB pathway. The findings show that the 13.5 d spaceflight may enhance the intestinal barrier-protective effect of BL.