This study explores the effects of KRAS and BRAF mutations on the heterogeneity, distribution, and uptake of extracellular vesicles (EVs), with a focus on their role in cancer biology and therapeutic potential. Nine cancer cell lines, including colorectal (Caco-2, HT29, WiDr, HCT116, LoVo) and lung cancer (H292, H1703, A549, H358), were selected based on KRAS and BRAF mutation status. EVs were isolated and characterized by TEM, Western blotting, and NTA. Proteomic analysis revealed that KRAS and BRAF mutant EVs were enriched in specific proteins, such as CD44 and LSR in KRAS mutations, and ITGB4 and CSPG4 in BRAF mutations. EV internalization was influenced by endocytosis and macropinocytosis, with competitive inhibition experiments showing the involvement of hyaluronic acid for CD44-mediated uptake and chondroitin sulfate for CSPG4. Finally, Doxorubicin-loaded KRAS-mutant EVs effectively delivered the drug to the nucleus, escaping lysosomal degradation. These findings suggest mutation-specific differences in EV biology and highlight their potential as targeted drug delivery vehicles in cancer therapy.