MHC class II molecules, in addition to their essential role as antigen-presenting molecules to CD4+ T cell receptor, have a signal-transducing role related to B cell function. We identified pro-IL-16 as one of the proteins associated with MHC class II-mediated signalling in an analysis of MHC class II-associated molecules using immunoprecipitation and proteomics data obtained from the 38B9 resting B cell line, and investigated the role of pro-IL-16 in resting B cell activation. We found that pro-IL-16, rather than mature form of IL-16, is present both in the cytoplasm and nucleus of resting B cells, and its expression is influenced by MHC class II-mediated signalling. In addition, overexpression of pro-IL-16 impaired resting B cell proliferation and this inhibitory effect was mediated through regulating nuclear factor (NF)-κB activation. Knock-down of pro-IL-16 expression using siRNA decreased the level of cell-cycle inhibitor p27kip and increased the level of Skp2. In addition, knock-down of pro-IL-16 modulated mitogen-activated protein kinase activation. Given that IL-16 acts as an immunomodulator by impairing antigen-induced T cell activation and its precursor, pro-IL-16, plays a role in regulating the cell cycle in T lymphocytes and T cell lymphoma, we concluded that pro-IL-16 is involved in resting B cell proliferation, similar to its function in T lymphocytes.