The present study was to investigate the mechanisms involved in macrophage activation by polysaccharides from the fruits of Rubus chingii Hu (RFPs). The results showed that RFPs enhanced pinocytic and phagocytic activity, promoted the expression and secretion of inflammatory factors (ROS, PTGS2, iNOS, IL-6, IL-10 and TNF-α) and chemokines (CCL2 and CXCL10), and boosted the expression of accessory and costimulatory molecules (CD40, CD80, CD86, MHC-I and MHC-II). RNA-Seq analysis identified 2564 DEGs, 1710 GO terms and 101 KEGG pathways. TNF was identified as the core gene via analysis of pathway information integration and PPI network. The western blot analysis combined with functional verification assay confirmed that MAPK, NF-κB and Jak-STAT pathways were essential to RFPs-mediated macrophage activation. TLR2 was revealed to be the functional receptor and involved in the early recognition of RFPs. These results indicated that RFPs modulated macrophage immune response mainly through TLR2-dependent MAPK, NF-κB and Jak-STAT pathways.