The present study was to elucidate the mechanisms underlying macrophage activation by total polysaccharides from Rhizoma Atractylodis Macrocephalae (RAMPtp). The results showed that RAMPtp significantly promoted productions of NO, ROS, cytokines and chemokines, enhanced pinocytic and phagocytic activity, and upregulated expressions of accessory and costimulatory molecules. RNA-seq analysis presented 2868 DEGs and 737 GO terms. PPI network analysis in combination with KEGG pathways as well as the western blot and functional verification assays indicated that NF-κB and STATs were the key regulators modulating the expressions of core gene TNF-α and IL-6 individually, and the transposition activation of NF-κB was identified as an early event in macrophage activation induced by RAMPtp. The involvements of MAPKs and PI3K-Akt pathways were also determined. These results indicated that immune response and immune function were regulated in RAMPtp-stimulated macrophages via a complex interaction network, in which NF-κB and Jak-STAT signaling pathways played a pivotal role.