Nanoparticles (NPs)-based adjuvants are attracting much attention in the development of vaccines. Previously, we reported a type of guanidyl-decorated polymeric NPs used as antigen delivery carriers for the first time. However, its un-degradability may restrict potential clinical translation. More importantly, the specific cellular pathway by which dendritic cells (DCs) endocytosed these NPs and the relationship among guanidyl with the antigen cross-presentation, cytokine secretion, and lymph node targeting still remain unclear. Here, we show NPs assembled by biodegradable methoxyl poly(ethylene glycol)-block-poly(ε-caprolactone)-graft-poly(2-(guanidyl) ethyl methacrylate) (mPEG-b-PCL-g-PGEM, PECG) copolymers can robustly activate DCs and promote their maturation; additionally antigen cross-presentation was improved both in vitro and in vivo. Significantly, our results also demonstrate the increase of surface guanidyl on nanoparticles modulates the depot effect and lymph node drainage of PECG NPs-based adjuvants, as well as immune responses, by regulating the secretion of cytokines including IFN-γ and TNF-α. Our study provides insights into the action of guanidyl-decorated nanoscale adjuvants and new adjuvants for vaccines containing protein antigens. We anticipate the strategy of guanidyl decoration to be a starting point for the development of more exciting immunoadjuvants.