Lymphocyte activation gene-3 (LAG-3; CD223) is a transmembrane protein that is structurally similar to CD4. Since LAG-3 has a much higher binding affinity to MHC class II than that of CD4, several approaches using soluble LAG-3 were used to modulate immune responses by activation or inhibition of MHC class II expressing antigen presenting cells. In this study, we constructed soluble pig LAG-3 containing a critical binding site (D1 and D2 region) to MHC class II molecules, combined with a constant region of an immunoglobulin (Ig) heavy chain. Flow cytometry analyses indicated that soluble pig LAG-3 binds to both pig and human MHC class II molecules. Moreover, soluble pig LAG-3 can inhibit human lymphocyte proliferation in the human-pig xenogeneic mixed lymphocyte reaction in a dose-dependent manner. These results indicate that soluble pig LAG-3 may be useful for controlling the xenogeneic T cell immune responses between the human and pig.