Polysaccharides from Physalis alkekengi L. have been proven to possess many biological activities. In our previous study, a homogeneous polysaccharide (PPSB) was extracted and purified from the fruits of Physalis alkekengi L., and the structure characterization was analyzed. The present study aimed to investigate the effects of PPSB on RAW264.7 macrophage cells activation and the underlying molecular mechanism. PPSB could activate RAW264.7 cells by not only enhancing the pinocytic and phagocytic activity, but also promoting the production of NO, ROS, TNF-α, and IL-6 in RAW264.7 cells. Meanwhile, PPSB up-regulated the expression of major histocompatibility complex (MHC-I/II) and costimulatory molecules such as CD40, CD80 and CD86. Mechanism studies showed that PPSB induced the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways. Moreover, the production of NO, TNF-α and IL-6 induced by PPSB in RAW264.7 cells were suppressed by specific MAPKs and NF-κB inhibitors. Further experiments with blocking antibodies demonstrated that the releases of NO, TNF-α and IL-6 and the activation of MAPKs and NF-κB induced by PPSB were decreased after TLR2 and TLR4 were blocked. Our date illustrated that PPSB was capable of activating the RAW264.7 cells via MAPKs and NF-κB signaling mediated by TLR2 and TLR4. •PPSB promoted the cell proliferation, pinocytosis and phagocytosis, and the production of NO, ROS and cytokines in RAW264.7.•PPSB upregulated the expressions of major histocompatibility complex and costimulatory molecules on RAW264.7 cells.•PPSB activated the MAPKs and NF-κB signaling pathways in RAW264.7 cells.•TLR2 and TLR4 were involved in PPSB-induced activation of RAW264.7 cells.