Icariin (ICA) and phosphorylated icariin (pICA) have excellent antiviral and antioxidant effects. However, whether ICA and pICA cause anti-LPS-induced intestinal damage remains unclear. In this study, we used Caco-2 cells as a model to investigate the protective effects of ICA and pICA on human colonic epithelial cells and explore their potential mechanisms. Our results indicated that ICA and pICA increased cell viability and decreased lactate dehydrogenase activity in Caco-2 cells. ICA and pICA also attenuated LPS-induced changes in intestinal epithelial cell permeability and reduced the levels of oxidative stress indicators, such as reactive oxygen species, malondialdehyde, and hydrogen peroxide, in Caco-2 cells. Antioxidant indicators, such as superoxide dismutase, glutathione peroxidase, catalase and total antioxidant capacity, were increased, while the levels of IL-1β, IL-6, IL-8 and TNF-α were reduced in the ICA and pICA groups. Furthermore, ICA and pICA decreased the gene abundance and enzyme activities of caspase-3, -8, -9 and -10 in Caco-2 cells. Our data suggest that ICA and pICA effectively attenuated LPS-induced changes in the oxidative stress, inflammation, apoptosis and intestinal permeability of intestinal epithelial cells. These findings provide new insight for treating LPS-induced intestinal injury.