The traditional use of Cordyceps militaris, an entomopathogenic fungus, in East Asian medicine has been well documented. Our previous study revealed that the fruiting body powder of C. militaris, referred to as Ryukyu-kaso, contains 1,3-β-glucan and stimulates bone marrow-derived dendritic cells via a dectin-1-dependent pathway. However, the immunomodulatory effects of soluble 1,3-β-glucan in Ryukyu-kaso and the underlying mechanisms remain unclear. In the present study, we aimed to investigate the immunostimulatory effects of the hot water extract of C. militaris fruiting body (RK-HWE) on bone marrow-derived dendritic cells and the involvement of the β-glucan receptor dectin-1. Our findings revealed that the hot water extract of C. militaris fruiting body contains soluble 1,3-β-glucan and potently induces bone marrow-derived dendritic cells to secrete both pro-inflammatory and immunoregulatory cytokines. Compared with the control, RK-HWE significantly increased the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, interfer-on-в, IL-12p70, and granulocyte-macrophage colony-stimulating factor (all ,i>p < 0.05), with TNF-α secretion increasing from 7.82 ± 1.57 (control) to 403.7 ± 59.7 pg/mL with (RK-HWE; p < 0.001). Notably, these immunostimulatory effects of RK-HWE were completely abolished in bone marrow-derived dendritic cells derived from dectin-1-knockout mice (p < 0.001, all cytokines), suggesting that dectin-1 is essential for immunomodulation induced by RK-HWE. These findings provide novel insights into the mechanisms underlying the immunostimulatory effects of RK-HWE and highlight its potential as a natural immunomodulatory agent for various therapeutic applications.