Major histocompatibility complex class II (MHC II) molecules are mainly expressed on antigen presentation cells and play an important role in immune response. It has been reported that MHC II molecules are also detected in serum as a soluble form (sMHC II molecules), and they are considered to be involved in the maintenance of self-tolerance. However, the mechanism by which sMHC II molecules are produced remains unclear. Invariant chain (Ii), also called CD74, plays an important role in antigen presentation of MHC II molecules. In the present study, we analyzed the role of Ii on the production of sMHC II molecules. We found that the amount of sMHC II molecules in serum was decreased in Ii-deficient mice compared to wild-type mice. sMHC II molecules were secreted from cells transfected with MHC II molecules and Ii but not from cells transfected with MHC II molecules alone. Moreover, isoform p41 of Ii-transfected cells induced more sMHC II molecules compared to isoform p31-transfected cells. The molecular weight of sMHC II molecules from MHC II and Ii p41-transfected cells was approximately 60 kDa, indicating that sMHC II molecules are a single heterodimer of α and β chains that is not associated with micro-vesicles. From the analysis of Ii-deletion mutants, we found that the luminal domain of Ii p41 is crucial for the production of sMHC II molecules. These results suggested that Ii has an important role in production of sMHC II molecules.