Objective Polyethylene glycol (PEG) is a frequently used osmotic laxative that accelerates gastrointestinal transit. It has remained unclear however, if PEG affects intestinal functions. We aimed to determine the effect of PEG treatment on intestinal sterol metabolism. Design Rats were treated with PEG in drinking water (7%) for two weeks or left untreated (controls). We studied the enterohepatic circulation of the major bile salt (BS) cholate with a plasma stable isotope dilution technique and determined BS profiles and concentrations in bile, intestinal lumen contents and feces. We determined the fecal excretion of cholesterol plus its intestinally formed metabolites. Finally, we determined the cytolytic activity of fecal water (a surrogate marker of colorectal cancer risk) and the amount and composition of fecal microbiota. Results Compared with control rats, PEG treatment increased the pool size (+51%; p<0.01) and decreased the fractional turnover of cholate (-32%; p<0.01). PEG did not affect the cholate synthesis rate, corresponding with an unaffected fecal primary BS excretion. PEG reduced fecal excretion of secondary BS and of cholesterol metabolites (each p<0.01). PEG decreased the cytolytic activity of fecal water (54 [46-62] vs. 87 [85-92] % erythrocyte potassium release in PEG treated and control rats, respectively; p<0.01). PEG treatment increased the contribution of Verrucomicrobia (p<0.01) and decreased that of Firmicutes (p<0.01) in fecal flora. Conclusion PEG treatment changes the intestinal bacterial composition, decreases the bacterial dehydroxylation of primary bile salts and the metabolism of cholesterol and increases the pool size of the primary BS cholate in rats.