Purpose <p>Mangiferin, a naturally occurring polyphenol, possesses remarkable antioxidant and anticancer properties. However, its therapeutic potential is hindered by its low and variable bioavailability. The present research work deals with the formulation and evaluation of mangiferin-loaded niosomes for breast cancer treatment, optimization using 3² factorial design, with particle size and entrapment efficiency as key responses.</p> Methods <p>All the batches of niosomes were prepared using thin film hydration methods and particle size, entrapment efficiency and cumulative drug release were determined. The optimized batch (F9) was characterized for SEM, XRD, FTIR, DSC, ex vivo permeability, in vivo pharmacokinetics and cytotoxicity.</p> Results <p>The F9 batch showed niosomal size of 221&#xa0;nm, zeta potential of -40.0 mV, entrapment efficiency of 89.5%, PDI of 0.227. Percent cumulative drug release in pH 6.8 medium revealed that 80.65% of the drug was released within 24&#xa0;h. Ex vivo intestinal permeability study indicated better permeation of mangiferin- loaded niosomes than pure drug. In vivo pharmacokinetic studies via the oral route indicated a 6.66-fold increase in Cmax and a 9.67-fold increase in AUC for mangiferin-loaded niosomes compared to pure drugs. Mangiferin-loaded niosomes were found to be 10.53 times more cytotoxic than pure drug on MCF-7 cell lines.</p> Conclusion <p>The findings suggest that mangiferin-loaded niosomes significantly enhance the anticancer activity of mangiferin highlighting their potential as an effective approach for breast cancer treatment.</p>