Osteosarcoma often occurs in children and adolescents with high invasiveness and high mortality. Polo‐like kinase 1 (PLK1) overexpressed in most tumors promotes cancer cell proliferation and transformation. PLK1 is considered as a therapeutic target for osteosarcoma. RNA interference‐based therapies are employed to combat osteosarcoma through silencing PLK1 gene expression. However, the treatment results remain unsatisfactory due to the lack of a safe and efficient nonviral gene vector. To tackle this hurdle, biodegradable and CO2‐derivative cationic poly(vinylcyclohexene carbonates) (CPCHCs) are used as gene vectors to perform a siPLK1 therapeutic strategy for osteosarcoma treatment. Of those CPCHCs, CPCHC60 demonstrates the most excellent performance in gene transfection efficiency, endo‐lysosome escaping, biodegradability, and biosafety. With the treatment of CPCHCs/siRNA nanoparticles, the expression level of PLK1 gene in osteosarcoma cells is significantly down‐regulated. Subsequently, cells are arrested in the G2/M phase and subsequently dead in the form of apoptosis, resulting in significant tumor regression both in vitro and in vivo. This study brings a new insight into the development of superior nonviral gene vectors for practical cancer treatment. Based on the results, the resulting nanoparticle‐based gene drug formation is considered to have a highly successful chance in further translational nanomedicine applications.