Abstract Fucoidan is a series of sulfated polysaccharides derived from brown algae that mainly consist of L-fucose. Fucoidan have various biological activities, and our investigation with experimental animal models and a healthy human trial demonstrated fucoidan from Cladosiphon okamuranus and Undaria pinnatifida effectively augmented anti-tumor immunity in combination with agaricus mycelium extract. In this context, we have reported that feeding of the fucoidan-agaricus mix (FAM) protected mice from immunosuppressive effect of 5-fluorouracil (5-FU). In current study, we confirmed dose-dependent immunoprotective effects of the dietary FAM (at 20 and 40 mg/day) by observing recovery in spleen weight, NK cell activity and IFN-γ production in 5-FU-treated mice. Furthermore, we investigated in vitro effects of FAM against 5-FU on growth and function of primary cultured immune-related cells. As the results with thioglycollate-induced peritoneal exudates cells (PEC), 5-FU dose-dependently suppressed the growth whereas FAM exerted no effects. However, production of cytokines such as IL-12p40 and TNF-α in PEC were stimulated by FAM treatment even in the presence of 5-FU. In the case of bone marrow derived cells differentiated with GM-CSF and M-CSF, FAM markedly enhanced the growth and IL-12 production of these cells. From these results, it was suggested that FAM contributed to amelioration of decline in anti-tumor immune function by 5-FU treatment through activation of innate immune cells.