Protamine is a promising marine-derived bioactive compound that is highly arginine-rich and has demonstrated unique advantages in medical and biological research. This study, for the first time, investigates the molecular mechanisms underlying the immunomodulatory effects of Salmon Protamine Sulfate (SPS), Protamine (SOP), and its polyethylene glycol (PEG) derivative (SOP-PEG) on RAW264.7 macrophages. The results demonstrate that both SOP and SOP-PEG significantly enhance the proliferation of RAW264.7 cells by promoting the secretion of pro-inflammatory cytokines and nitric oxide (NO), increasing ROS production, and improving antioxidant capacity, in comparison to SPS. Elevated ROS levels play a crucial role in enhancing macrophage immune activity, while the enhanced antioxidant defense mechanisms help maintain redox homeostasis and protect against oxidative stress-induced cellular damage. A Western blot analysis reveals that SOP and SOP-PEG notably regulate the expression of key proteins associated with the PI3K/Akt signaling pathway and anti-apoptotic mechanisms. Furthermore, a flow cytometry analysis indicates a significant increase in the G2/M-phase cell population in the treatment groups, which is corroborated by Western blot data showing alterations in critical regulatory proteins. Notably, SOP-PEG exhibits the strongest effects in regulating macrophage immune activity, which can be attributed to the enhanced stability and prolonged bioactivity resulting from the PEGylation of SOP. This comprehensive study reveals how SOP and SOP-PEG enhance macrophage immune function through multiple mechanisms, including PI3K/Akt activation, redox regulation, and cell cycle modulation. It provides valuable insights and a theoretical foundation for their potential applications in immunotherapy and immune regulation.