Propranolol (PROP) undergoes extensive first-pass metabolism by the liver resulting in a relatively low bioavailability (13–23%); thus, multiple oral doses are required to achieve therapeutic effect. Since some studies have reported that glucosamine (GlcN) can increase the bioavailability of some drugs, therefore, it is aimed to study whether GlcN can change the pharmacokinetic parameters of PROP, thus modulating its bioavailability. When PROP was orally co-administered with GlcN (200 mg/kg) to rats, PROP area under curve (AUC) and maximum concentration (Cmax) were significantly decreased by 43% (p < 0.01) and 33% (p < 0.05), respectively. In line with the in vivo results, in silico simulations confirmed that GlcN decreased rat intestinal effective permeability (Peff) and increased PROP clearance by 50%. However, in situ single pass intestinal perfusion (SPIP) experiments showed that GlcN significantly increased PROP serum levels (p < 0.05). Furthermore, GlcN decreased PROP disposition/distribution into cultured hepatocytes in concentration dependent manner. Such change in the interaction pattern between GlcN and PROP might be attributed to the environment of the physiological buffer used in the in vitro experiments (pH 7.2) versus the oral administration and thus, enhanced PROP permeability. Nevertheless, such enhancement was not detected when everted gut sacks were incubated with both drugs at the same pH in vitro. In conclusion, GlcN decreased PROP serum levels in rats in a dose-dependent manner. Such interaction might be attributed to decreased intestinal permeability and enhanced clearance of PROP in the presence of GlcN. Further investigations are still warranted to explain the in vitro inhibitory action of GlcN on PROP hepatocytes disposition and the involvement of GlcN in the intestinal and hepatic metabolizing enzymes of PROP at different experimental conditions.