Immunostimulants (IS) are considered a promising approach for improving resistance to pathogens in fish aquaculture. At present, development of IS are complicated due to limited knowledge on the mechanisms of their action. To assess the use of global gene expression analysis for screening of candidate IS we applied lentinan, a beta-glucan from the mushroom Lentinula edodes, as a model. After feeding rainbow trout (Oncorhynchus mykiss) with lentinan-supplemented (L) and control (C) diets for 37 days, fish were injected with bacterial lipopolysaccharide (LPS), a classical inducer of inflammation. Gene expression was analyzed in LPS-challenged compared to saline-injected fish using a salmonid 1.8k cDNA microarray (SFA2.0 immunochip) and real-time qPCR. Spleen was selected for data analyses due to highest magnitude of responses and its key role in the fish immune system. A group of genes implicated in acute inflammatory responses was higher induced in C versus L, including IFN-related and TNF-dependent genes (galectins and receptors, signal transducers and transcription factors), genes involved in MHC class I antigen presentation and leukocyte recruitment. A similar trend was observed in metabolism of iron and xenobiotics, markers of oxidative and cellular stress. Interestingly, differences between C and L were similar to those observed between salmon with low and high resistance to infectious salmon anemia virus. Genes with equal responses to LPS in L and C were related to cell communication (cytokines, chemokines and receptors), signal transduction, activation of immune cells, apoptosis, cellular maintenance and energy metabolism. In conclusion, lentinan decreased the expression of genes involved in acute inflammatory reactions to the inflammatory agent while major parts of the immune response remained unchanged. Such effects are expected for IS, which should modify immunity by enhancing beneficial and reducing detrimental responses.