The purpose of this study was to evaluate the obesity preventing or relieving effect of insoluble dietary fiber (IDF) from Pleurotus eryngii through its modulations on intestinal microbiota. The IDF was extracted and structurally characterized through Fourier transform infrared spectroscopy (FT-IR), X-ray and thermogravimetric (TG) analysis. The mice for in vivo experiments were divided into obesity preventing group (feeding high-fat diet while gavage IDF simultaneously) and obesity relieving group (IDF intervention after the establishment of the obese model) to evaluate its hypolipidemic effect. The yield of P. eryngii IDF extracts was 56.49% (on P. eryngii dry weight base) with IDF content of 85.15% in the extracts. The in vivo results showed that the body weight of the obesity-preventing groups was still increasing after intervention with IDF, but the growth rate was lowered, and the abnormal lipid metabolism and inflammation syndromes were alleviated; the acetic acid content of the medium and low dose dietary fiber intervention groups was significantly higher than that of the normal group, and the structure of the intestinal flora was improved. The mice weight from the obesity-relieving group decreased significantly. The abnormal lipid metabolism and inflammation were alleviated. In conclusion, The IDF intervention decreased the abnormal lipid metabolism and inflammation in both obesity-preventing and relieving experiments. The intestinal flora structure was obviously improved. P. eryngii IDF is a good candidate as hypolipidemic agent.