The notion that certain formerly regarded “inert” pharmaceutical excipients are capable of modifying the bioavailability of oral drugs has gained increasing attention in recent years. For instance, the commonly-used solubilizing agent polyethylene glycol 400 (PEG 400) exhibits a sex-specific effect on the bioavailability of ranitidine in both humans and rats, mediated by the efflux transporter P-glycoprotein (P-gp). To determine whether such in vivo effect could be predicted by in vitro tests, an in vitro/ex vivo model was established using tissues from male and female rats to characterize the influence of PEG 400 on the intestinal transport of ranitidine in the absence and/or presence of a Pgp inhibitor, cyclosporine A (CsA). We found the absorptive permeability of ranitidine in the small intestine (duodenum, jejunum, ileum) and colon was higher in females compared with males. PEG 400 significantly increased the absorption and decreased the secretion of ranitidine in the intestine of male rats (p < 0.05), but no such effects were observed in female intestines. In addition, while the P-gp inhibitor CsA increased the intestinal uptake of ranitidine in both male and female rats, a greater extent of intestinal transport modulation was observed in males compared to females. These in vitro data on the influence of PEG 400 on the intestinal transport of ranitidine in a sex-dependent manner are in agreement with previously published in vivo data. This good in vivo-in vitro correlation means that the in vitro method will be quicker, cheaper and easier to investigate any sex-related influence of pharmaceutical excipients on oral drug bioavailability.