Porphyra yezoensis is a red alga consumed mainly in Korea, Japan, and China for food. This study researches the immunological effect of pepsin extract of P. yezoensis (PPEE) on murine splenocytes. PPEE was not toxic on murine splenocytes and dramatically increased the proliferation of splenocytes compared with untreated control. Flow cytometry assay performed to sum up the effect of PPEE (31.3 and 62.5 µg mL-1 ) on major immune cells revealed that PPEE had no effect on the function of CD3e+ CD4+ T-helper cells, CD3e+ CD8+ T-cytotoxic cells, or CD44+ CD62L- effector T cells in splenocytes compared with untreated control. More importantly, CD45+ CD11b+ macrophage and dendritic cell populations and Ly-6C+ Ly-6G+ macrophages/monocytes in splenocytes were activated by PPEE treatment compared with untreated control. Further experiments showed that PPEE treatment increased the secretion of macrophage-derived cytokines such as interleukin-1β, tumor necrosis factor-α, and interleukin-12, and macrophage-activating cytokines interferon-γ and interleukin-10 compared with untreated control. Taken together, these results suggest that PPEE has an immune stimulatory effect on macrophages, dendritic cells, and memory T cells. This property signifies the potential medicinal value of PPEE in clinical implications for immune-compromised diseases. © 2017 Society of Chemical Industry.