Sargassum horneri (SH) is a seaweed that has several features that benefit health. In this study, we investigated the immune-enhancing effect of SH, focusing on the role of spleen-mediated immune functions. Chromatographic analysis of SH identified six types of monosaccharide contents, including mannose, rhamnose glucose, galactose xylose and fucose. SH increased cell proliferation of primary cultured naïve splenocytes treated with or without cyclophosphamide (CPA), an immunosuppression agent. SH also reversed the CPA-induced decrease in Th1 cytokines. In vivo investigation revealed that SH administration can increase the tissue weight of major immune organs, such as the spleen and thymus. A similar effect was observed in CPA-injected immunosuppressed BALB/c mice. SH treatment increased the weight of the spleen and thymus, blood immune cell count and Th1 cytokine expression. Additionally, the YAC-1-targeting activities of natural killer cells, which are important in innate immunity, were upregulated upon SH treatment. Overall, our study demonstrates the immune-enhancing effect of SH, suggesting its potential as a medicinal or therapeutic agent for pathologic conditions involving immunosuppression.