Ganoderma lucidum is a medicinal mushroom in China and other Asian countries. The polysaccharide from G. lucidum (PS-G) is a branched (1-->6)-beta-d-glucan moiety. In this study, we examined the effects of PS-G on human monocyte-derived dendritic cells (DCs) with microarray analysis by Human Genome U133 Plus 2.0 GeneChip. In comparing mean signal values between PS-G-treated DCs with untreated DCs, 3477 (17%) probe sets were up-regulated, and 4418 (19%) probe sets were down-regulated after PS-G treatment. These results demonstrate that genes associated with phagocytosis (CD36, CD206, and CD209) are decreased and genes associated with proinflammatory chemokines (CCL20, CCL5, and CCL19), cytokines [interleukin (IL)-27, IL-23A, IL-12A, and IL-12B], and costimulatory molecules (CD40, CD54, CD80, and CD86) are increased. To confirm the microarray data, we further investigated the effect of PS-G on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with ovalbumin (OVA)/PS-G showed that the anti-OVA IgG2a levels were significantly increased compared with OVA alone in BALB/c mice. Together, our data demonstrate that PS-G could effectively promote the activation and maturation of immature DCs, preferring a T helper 1 response. Furthermore, the results also demonstrate that the data from microarray analysis could be correlated with the in vivo effect of the immune-enhancing compound.