The synthesis of a novel class of polymer prodrug nanoparticles with anticancer activity is reported by using squalene, a naturally occurring isoprenoid, as a building block by the reversible addition-fragmentation (RAFT) technique. The RAFT agent was functionalized by gemcitabine (Gem) as anticancer drug, and the polymerization of squalenyl-methacrylate (SqMA) led to well-defined macromolecular prodrugs comprising one Gem at the extremity of each polymer chain. The amphiphilic nature of the resulting Gem-PSqMA conjugates allowed them to self-assemble into long-term stable and narrowly dispersed nanoparticles with significant anticancer activity in vitro on various cancer cell lines. To confer stealth properties on these nanoparticles, their PEGylation was successfully performed, as confirmed by X-ray photoelectron spectroscopy (XPS) and complement activation assay. It was also shown that the PEGylated nanoparticles could be internalized in cancer cells to a greater extent than their non-PEGylated counterparts.