Porcine circovirus type 2 (PCV2)-associated diseases have led to great economic losses to the pig industry. Our lab previously found that conjugation of chitosan oligosaccharides (COS) or via a carrier protein enhanced the immunogenicity of PCV2 vaccine against infectious pathogens. However, precise mechanisms and signal transduction pathways underlying the efficacy of COS conjugation remains poorly defined. In this study, to better understand the effects and mechanism of COS conjugates maintain the adjuvant potential in vivo, we investigated its augmentation of macrophage function, including cell activation, NO production, cytokine production and phagocytosis. Additionally, the role of Toll-like receptors (TLR) proteins in this process was also assessed. The results indicate that, as compared to the PCV and PCV/COS, conjugation of COS effectively enhanced the NO production, cytokines generation and phagocytosis activity of macrophages. Noticeably, the generation of NO and proinflammatory cytokines was closely related to the TLR2/4 signaling pathways, strongly suggesting that conjugation of COS regulates innate and adaptive immunity by activation of macrophages, resulting in immune enhancement. In summary, the present study provides a potential mechanism of COS conjugation as a novel adjuvant to improve immune responses against various diseases.