Hemophilia B, caused by factor IX (FIX) deficiency, remains the best candidate for mRNA-based gene therapy. However, efficient hepatic delivery of mRNA continues to be a significant challenge. In this study, we developed and evaluated berberine-functionalized cationic liposomes as a novel delivery platform for FIX mutant mRNAs. Berberine incorporation enhanced the liposome’s membrane fluidity and fusion potential, facilitating improved intracellular delivery and endosomal escape. Six different FIX variants were designed, transcribed, and screened for optimal expression. Our results demonstrate that berberine liposomes significantly enhance hepatocyte transfection and enable sustained FIX production, particularly with the TmL mutant. These findings suggest that berberine-functionalized liposomes represent a delivery strategy for mRNA therapeutics, leveraging the natural liver tropism common to lipid-based systems while aiming to enhance hepatocyte transfection efficiency.