This study was aimed to characterize Gracilaria lemaneiformis polysaccharides and evaluate their protective effects on Lipopolysaccharide-induced injury in IEC-6 cells. The G. lemaneiformis polysaccharide was degraded by UV/H2O2 treatment and purified to three fractions named GLP-1.0 M, GLP-1.4 M and GLP-1.6 M. The purified fractions were mainly composed of galactose, glucose and xylose. The structural analysis showed that GLP-1.6 M was a typical sulfated red alga polysaccharide containing the linear backbone of β-(1 → 3)- and α-(1 → 4)-linked galactosyl residues, anhydro-galactose units. In the Lipopolysaccharide-induced IEC-6 cells model, GLP-1.6 M exerted the strongest in vitroanti-inflammatory activity by inhibiting the release and expressions of tumor necrosis factor-α, interleukin-6 and interleukin-1β by 89.93%, 67.82% and 38.06%, respectively. Meanwhile, GLP-1.6 M enhanced the intestinal barrier function via up-regulating the expressions of tight junctions and mucin. Therefore, the purified polysaccharide from G. lemaneiformis could be a promising candidate for maintaining intestinal health in the food and pharmaceutical industries.