Rationale: Use of traditional anticancer chemotherapeutics has been hindered by the multifactorial nature of multi-drug resistance (MDR) development and metastasis. Recently, cationic polycarbonates were reported as novel unconventional anticancer agents that mitigated MDR and inhibited metastasis. The aim of this study is to explore structure-anticancer activity relationship. Specifically, a series of cationic guanidinium-based random copolymers of varying hydrophobicity was synthesized with a narrow polydispersity (Ð = 1.12-1.27) via organocatalytic ring-opening polymerization (OROP) of functional cyclic carbonate monomers, and evaluated for anticancer activity, killing kinetics, degradability and functional mechanism. Methods: Linear, branched and aromatic hydrophobic side chain units, such as ethyl, benzyl, butyl, isobutyl and hexyl moieties were explored as comonomer units for modulating anticancer activity. As hydrophobicity/hydrophilicity balance of the polymers determines their anticancer efficacy, the feed ratio between the two monomers was varied to tune their hydrophobicity. Results: Notably, incorporating the hexyl moiety greatly enhanced anticancer efficiency and killing kinetics on cancer cells. Degradation studies showed that the polymers degraded completely within 4-6 days. Flow cytometry and lactate dehydrogenase (LDH) release analyses demonstrated that anticancer mechanism of the copolymers containing a hydrophobic co-monomer was concentration dependent, apoptosis at IC50, and both apoptosis and necrosis at 2 × IC50. In contrast, the homopolymer without a hydrophobic comonomer killed cancer cells predominantly via apoptotic mechanism. Conclusion: The hydrophobicity of the polymers played an important role in anticancer efficacy, killing kinetics and anticancer mechanism. This study provides valuable insights into designing novel anticancer agents utilizing polymers.