Two immunomodulatory polysaccharides (Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cells. Both the level of released nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) mRNA were significantly enhanced in the RAW264.7 macrophages cells treated by Vp2a-Ⅱ and Vp3. Vp2a-Ⅱ (100–800 µg/mL) and Vp3 (400 µg/mL) could significantly increase the phagocytic activity of RAW264.7 cells and the secretion and mRNA expression of TNF-α and IL-6 in a concentration-dependent manner through affecting mitogen-activated protein kinase (MAPK) activity and nuclear factor κB (NF-κB) nuclear translocation. Vp2a-Ⅱ might activate the MAPK signaling pathways and induce the nuclear translocation of NF-κB p65, whilst Vp3 likely activated the NF-κB and MAPK signaling pathways without influencing the p38 MAPK route.