BackgroundThere are few studies about the differences in the composition of moisture, ash, crude protein, crude fat, crude polysaccharide and ergothioneine in Ganoderma lucidum spore powder (GLSP) from different origins. As for GLSP after oil extraction (OE‐GLSP), there are still lots of bioactive substance in it. It can be seen that OE‐GLSP has certain biological activity. The effect of OE‐GLSP on the improvement of intestinal barrier function has been less studied.ResultsThe results showed that there were significant differences for GLSP from five different origins (Anhui, Jilin, Jiangxi, Shandong and Zhejiang) in moisture (0.065–0.113%), ash (0.603–0.955%), crude fat (42.444–44.773%), crude polysaccharide (2.977–4.127%), crude protein (14.761–17.639%) and ergothioneine (0.552–1.816 mg g−1) (P < 0.05). The monosaccharides of GLSP polysaccharide mainly consist of glucose, galactose, mannose, rhamnose, etc. Moreover, the effects of OE‐GLSP supplementation on the regulation of organ index, colonic tissue and intestinal microbiota in C57BL/6J mice were investigated. The supplement of OE‐GLSP could restore the organ index and weight loss of antibiotic‐treated mice. Moreover, OE‐GLSP led to the improvement of intestinal dysbiosis by enriching Bacteroidetes, Firmicutes, Lactobacillus and Roseburia, and increasing the Firmicutes/Bacteroidetes ratio. In addition, OE‐GLSP intervention repaired intestinal barrier dysfunction by increasing the expression of tight junction proteins (Occludin, Claudin‐1 and E‐cadherin).ConclusionDifferent GLSP from five origins exhibited significant differences in microstructure and contents of crude polysaccharide, crude protein, crude fat, water, ash and ergothioneine. Moreover, it was found that OE‐GLSP could improve the intestinal barrier function and induce potentially beneficial changes in intestinal flora. © 2024 Society of Chemical Industry.