Polysaccharides from edible mushrooms possess important immunomodulating effects on immune cells including monocytes and macrophages. Macrophages activated by LPS/IFNγ are polarized toward inflammatory macrophages, whereas the anti-inflammatory properties of alternative activated macrophages play an important regulatory role in the innate immune system. We here show that the Pleurotus citrinopileatus mushroom polysaccharide (PCPS) can modulate the monocyte-to-macrophage differentiation early at the monocyte stage. Using both human THP-1 monocytic cells as well as human peripheral monocytes, we showed that PCPS inhibits the secreted levels of the pro-inflammatory cytokines TNF and IL-6, after stimulation of macrophages derived from PCPS-treated monocytes, with IFNγ + LPS. In addition, the glucan induced a tendency to increase the secreted levels of the anti-inflammatory cytokine IL-10, enhanced the expression levels of CCL2 and CCL8 mRNAs, and inhibited expression of CCR2 mRNA in the IFNγ/LPS activated macrophages. Interestingly, these data suggest that PCPS can induce a long-lasting anti-inflammatory effect in monocytes. Treatment of monocytes with laminarin and antibodies against Dectin-1 and TLR2 during PCPS treatment affected the glucan-modulated macrophage differentiation. In summary, the results of this study indicate that the glucan directs the differentiation of monocytes toward a macrophage cell population with reduced pro-inflammatory capacity via Dectin-1 and TLR2.