Carnosic acid (CA) is a natural phenolic compound with several biomedical actions. This work was performed to study the use of CA-loaded polymeric nanoparticles to improve the antitumor activity of breast cancer cells (MCF-7) and colon cancer cells (Caco-2). CA was encapsulated in bovine serum albumin (BSA), chitosan (CH), and cellulose (CL) nanoparticles. The CA-loaded BSA nanoparticles (CA-BSA-NPs) revealed the most promising formula as it showed good loading capacity and the best release rate profile as the drug reached 80% after 10 h. The physicochemical characterization of the CA-BSA-NPs and empty carrier (BSA-NPs) was performed by the particle size distribution analysis, transmission electron microscopy (TEM), and zeta potential. The antitumor activity of the CA-BSA-NPs was evaluated by measuring cell viability, apoptosis rate, and gene expression of GCLC, COX-2, and BCL-2 in MCF-7 and Caco-2. The cytotoxicity assay (MTT) showed elevated antitumor activity of CA-BSA-NPs against MCF-7 and Caco-2 compared to free CA and BSA-NPs. Moreover, apoptosis test data showed an arrest of the Caco-2 cells at G2/M (10.84%) and the MCF-7 cells at G2/M (4.73%) in the CA-BSA-NPs treatment. RT-PCR-based gene expression analysis showed an upregulation of the GCLC gene and downregulation of the BCL-2 and COX-2 genes in cells treated with CA-BSA-NPs compared to untreated cells. In conclusion, CA-BSA-NPs has been introduced as a promising formula for treating breast and colorectal cancer.