Side effects of chemotherapy are burning questions for physicians and patients involved in cancers. Ganoderma lucidum is a widely consumed traditional Chinese medicine and edible mushroom with multiple functional properties. The present study aims to investigate the potential of polysaccharides from spore of G. lucidum (SGP) on small intestinal barrier function recovery against paclitaxel (PTX) challenge in a breast cancer mice model and IEC-6 cell line. The 4T1 tumor-bearing mice were treated with PTX together with four-week daily oral administration of SGP. Results indicated that combination of PTX and SGP reversed body weight lost and remolded the histology of small intestine, accompanied with promoted proliferation but suppressed apoptosis in intestinal cells. Intestinal barrier function was enhanced by the combination as indicated by reduced endotoxemia and the up-regulation of tight junction proteins, including Zonula occludens-1 (ZO-1), E-cadherin, β-catenin and Occludin. The protection of SGP was further confirmed in IEC-6 cells affected by PTX in vitro. The combination treatment prevented PTX-induced apoptosis in IEC-6 by inhibiting microtubule polymerization, and the aforementioned tight junction proteins were also upregulated. These findings suggest a promising protective effect of SGP against small intestinal barrier injury caused by PTX, highlighting its clinical implication against the chemotherapy side effects.