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An alkali-extracted polysaccharide (PCAPS1) was isolated and purified from the Poria cocos. Our results proved that PCAPS1 was a neutral polysaccharide with a molecular weight of 11.5 kDa. The monosaccharide composition, methylation and NMR analysis results displayed that the polysaccharide was mostly comprised of β-1,3-glucan with 1,4 and 1,6 branches. The Immune activity and mechanism of PCAPS1 were evaluated in RAW264.7 cells. The enzyme-linked immunosorbent assay (ELISA) analysis revealed that PCAPS1 increased the tumor necrosis factor-α (TNF-α) secretion. RNA-sequencing data analysis suggested that PCAPS1 activated macrophages by the classic NF-κB pathway. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis confirmed that PCAPS1 enhanced mRNA expression levels of TNF-α and nuclear factor κB (NF-κB) in RAW264.7 cells. Simultaneously, the fluorescence nuclear transport experiment showed that PCAPS1 activated RAW264.7 cells by inducing the NF-κB p65 translocation. Our results indicated that PCAPS1-induced TNF-α expression was mediated via the NF-κB signaling pathway.