The immunomodulatory effects of R-phycoerythrin (R-PE) from Porphyra haitanensis were investigated by a hydrocortisone (HC)-induced immunosuppressive model in the present research. The results showed that R-PE had immunomodulatory potential, such as increasing spleen and thymus indexes, ameliorating spleen morphology, enhancing the phagocytic capability of macrophages, promoting spleen T lymphocyte proliferation, and strengthening serum hemolysin formation. Furthermore, R-PE elevated the CD4+/CD8+ ratio and up-regulated the levels of cytokines for Th1, Th2, and Th17 and the mRNA expression of transcription factors T-bet, GATA3, and RORγt. Besides, the levels of cytokines for Treg and transcription factor Foxp3 were down-regulated. The levels of T-bet/GATA3 and RORγt/Foxp3 were increased. R-PE activated the phosphorylation of NF-κB through the TLR4-mediated differentiation pathway. These findings suggested that R-PE exerted immunomodulatory activities in the innate and adaptive immune systems through TLR4/NF-κB mediated CD4+ T cell activation and differentiation, which facilitated the further application of R-PE as an immunomodulator.