Berberine is a naturally occurring plant-derived alkaloid, isolated from Berberis species. Berberine possess multiple pharmacological properties including anti-inflammatory, Anti-oxidant, Anti-diabetic, Antihypertensive, Antidepressant, Antidiarrheal, Antimicrobial, Neuroprotective, Hepatoprotective, Antitumor and Anticancer activity. Though these outcomes are very promising, its low water solubility and oral bioavailability restrict its clinical uses. Hence, in the present study we have reported the entrapment of Berberine (BBR) in Bovine Serum Albumin Nanoparticle (BSA NPs) and its cytocompatibility to Breast Cancer cell line (MDA-MB-231). BSA NPs and Berberine loaded BSA nanoparticles (BBR-BSA NPs) were successfully synthesized by desolvation method. The BSA NPs alone and BBR-BSA NPs were extensively characterized for the size, shape, surface charge, morphology, stability, drug content and in vitro drug release. Physiochemical characterization of prepared nanoparticles was done by using DLS, FESEM, FTIR, DSC and TGA. The size of synthesized nanoparticles were found to be within the suitable range for drug delivery, having positive charge analysed by zeta potential. The SEM images of nanoparticles implied that prepared nanoparticles are of spherical shape. The drug entrapment efficiency of prepared nanoparticles was found to be 85 % with a drug loading capacity of 7.78 %. The FTIR spectra confirms the synthesis of Berberine loaded nanoparticles. Time dependent stability study suggests that nanoparticles are quite stable in aqueous solution at pH 7.4. The MTT assay, Trypan blue assay and AO-EtBr staining proved that the prepared NPs are selectively toxic towards breast cancer cell lines and kill the cells more efficiently as compared to Berberine alone. The results of intracellular uptake studies suggest that BBR-BSA NPs could effectively improve the anticancer activity of BBR by delivering it to target site for potential therapeutic use. This work provides a novel therapeutic approach for inducing breast cancer cell death or inhibiting cell growth. All the above information suggests that BBR-BSA NPs may emerge as a novel paradigm for treatment of breast cancer.