Background: Polyinosinic-polycytidylic acid (pIC) is a synthetic analog of double-stranded RNA. It is used as a synthetic adjuvant to induce an adaptive immune response. However, the effect of pIC on the development of mediastinal fat-associated lymphoid clusters (MFALCs) that regulate intrathoracic hemostasis has remained unidentified. Methods: We investigated the impact of intranasal (i.n.) administration (pIC i.n. group) and intravenous (i.v.) administration (pIC i.v. group) of pIC on both MFALCs and lung tissue. Results: Compared with the control phosphate-buffered saline (PBS) groups, both pIC-administered groups displayed a significant increase in the MFALC size (particularly in the pIC i.n. group), area of MFALC high endothelial venules (HEVs), area of lymphatic vessels (LVs), number of proliferating cells (particularly in the pIC i.v. group), and number of immune cells (B220+ B-lymphocytes, CD3+ T-lymphocytes, Iba1+ macrophages, and Gr-1+ granulocytes) in both MFALCs and lung tissues. In addition, a positive correlation was detected between MFALC size and proliferating cells, immune cell population, LVs, and HEVs within MFALCs in both groups. Except for the proliferating cell and B-lymphocyte populations in the i.n. administered group and granulocyte populations in both i.n. and i.v. administered routes, such correlations were significant. Conclusion: In all, our data indicate that local or systemic administration of pIC induces the development of MFALCs and can be used as an immunostimulant therapeutic strategy