Interactions of β-carotene in the absence or presence of stearylamine (SA, positive charge inducer) with liposomes as model membranes were characterized. The morphology of all liposomes was almost spherical in shape, well dispersed and less aggregated for empty and encapsulated vesicles. The mean particle size distribution of empty liposomal samples was clustered around 43.82 ± 21.29 nm. The presence of SA in liposomal membranes decreased the density of negative charge, resulting in a positive zeta potential. The introduction of β-carotene or β-carotene mixed with SA into liposomes exhibited a shift to higher temperature. FTIR study confirmed the interaction of β-carotene or β-carotene mixed with SA with the liposome’s moieties. In the absence of external gamma irradiation, the IC₅₀ value for free β-carotene in PC-3 cells was 29.24 µg/ml, whereas positively charged liposomal β-carotene showed an IC₅₀ of 34.74 µg/ml, indicating slightly greater cytotoxicity of the free form when used alone. However, under combined treatment with 5 Gy gamma irradiation, the IC₅₀ for positively charged liposomal β-carotene was markedly reduced to 11.66 µg/ml compared with 32.46 µg/ml for free β-carotene, demonstrating the superior cytotoxic effect of the charged liposomal formulation in the presence of irradiation. Flow cytometric analysis revealed that after 48 h of exposure to positively charged liposomal β-carotene. The treatment with the IC50 of positively charged liposomal β-carotene resulted in (58.14% and 33.67%) followed by liposomal β-carotene (51.52% and 43.66%) cell accumulation in the G0-G1 and S phases, respectively. The current findings demonstrated that natural product preparations could be a viable therapy option for prostate cancer in place of pharmaceutical therapies.