β-glucans are a commonly used immunostimulant/prebiotic in many aquaculture applications for boosting the immune status in fish. However, the method of action as an immunostimulant has not been fully deciphered. To determine the immunomodulatory effects of β-glucans on the innate immune response, we stimulated the rainbow trout spleen macrophage-like cell line (RTS11) with β-1,3/1,6-glucans for 4 h. This study uses a whole transcriptomic approach to analyse the immunomodulatory properties of β-glucans. Several proinflammatory pathways were found to be enriched after stimulation, demonstrating the immunomodulatory effects of β-glucan supplementation. Several pathways relating to responses to bacteria were also found to be enriched. This study clearly demonstrates the immunomodulatory effects of the supplementation of β-glucans within an aquaculture setting and further validates the use of cell lines as predictive models to interpret the responses caused by dietary intervention.