Various perspectives had been utilized to enhance the poor intestinal permeability and bioavailability of drugs with low water solubility. Berberine (Brb) is a unique molecule that possesses multiple therapeutic activities such as antimicrobial, anti-inflammatory, antioxidant and anti-hyperglycemic effects. To improve Brb permeability and bioavailability, this study presents a newly developed formulation, namely Brb hyaluronate-based liposomes, prepared by using film hydration method and characterized by dynamic light scattering measurements, entrapment efficiency percentage (EE%), transmission electron microscope (TEM), in vitro drug release and physical stability. The bioavailability of the selected formulations was assessed in vivo after oral administration to rats. The results revealed an enhanced effect of hyaluronic acid on the entrapment efficiency, reaching 78.1 ± 0.1% with mean size 520.7 ± 19.9 nm. Sustained release of Brb was recorded up to 24 h in comparison to Brb solution. Physical stability was maintained for three months at refrigeration temperature. Results of pharmacokinetics studies indicated the potential of the liposomal formulation to increase the oral bioavailability of Brb and to accelerate its entry into the bloodstream. The obtained results are accredited to the lipophilic nature of the prepared system, resembling the structural features of bio-membrane, in addition to their small size that enhances intestinal penetration.