Uncontrolled inflammation is the driver of numerous lung diseases. Current treatments, including corticosteroids and bronchodilators, can be effective. However, they often come with notable side effects. siRNA is a promising therapeutic modality for immune regulation. However, effective delivery of siRNA is challenged by issues related to cellular uptake and localization within tissues. This study investigates a series of guanidinium-functionalized polymers (Cn-Guan) designed to explore the effects of amphiphilicity on siRNA complexation and efficiency in vitro and in vivo. Nine polymers with varying side chain lengths (C3, C5, C7) and molecular weights (17 kDa, 30 kDa, 65 kDa) were synthesized, forming polyplexes with siRNA. Characterization revealed that C7-Guan/si_scr polymers exhibited the smallest polyplex sizes and the tightest complexation with siRNA. In vitro studies showed that 65 kDa polymers had the highest gene knockdown efficiency, with C3 and C5-Guan/si_TNF-α achieving ∼70 % knockdown, while C7-Guan/si_TNF-α achieved ∼30 %. In vivo, C7-Guan/Cy5-siRNA demonstrated the highest lung accumulation, and all polymers showed ∼70 % TNF-α knockdown with a low siRNA dosage (0.14 mg/kg) in a murine lung inflammation model. C7-Guan polymers, despite lower in vitro efficiency, were quite effective in vivo, potentially due to enhanced serum stability. These findings demonstrate that Cn-Guan/siRNA polyplexes are effective and safe for attenuating pulmonary inflammation and provide important insights for the development of future siRNA delivery vectors for lung disease treatment.