In this study, chestnut (Castanea sativa) shell was extracted with different solvents, and immunomodulatory activity was investigated in an in vitro model system using blood and intestinal leukocytes of Oncorhynchus mykiss. Gallic acid (GA) was used as a standard. Chestnut shell extract (CSE) and GA readily entered both blood and intestinal leukocytes. Superoxide anion production and phagocytosis were decreased by low doses of CSE and increased with high doses. CSE and GA differently regulated cytokine expression in blood and intestinal leukocytes. High doses of CSE upregulated IL-1β, TNF-α, and IL-10 in intestinal leukocytes and IL-10 in blood leukocytes. Low doses of CSE upregulated IL-1β and TNF-α in blood leukocytes. GA appeared to be effective only in blood leukocytes. The effects of CSE on pro- and anti-inflammatory cytokines seemed to suggest an alert effect of the immune defense system against a possible infectious agent. The less evident effect of GA in comparison to CSE could have been attributable to the synergistic and/or additive effects of polyphenols in the latter. The immune-stimulating activity of CSE reported here could be useful for future practical applications in fish health.