The transcription factor STAT6 plays an important role in cell responses to IL-4. Its activation is tightly regulated. STAT6 phosphorylation is associated with JAKs, whereas dephosphorylation is associated with specific phosphatases. Several studies indicate that proteases can also regulate STAT6. The aim of this study was to investigate the nature of these proteases in mouse T cell lines. We found that STAT6 was degraded in cell extracts by calcium-dependent proteases. This degradation was specifically prevented by calpain inhibitors, suggesting that STAT6 was a target for these proteases. This was supported by the cleavage of STAT6 by recombinant calpains. The proteolytic regulation of STAT6 was more complex in vivo. Calcium signaling was not sufficient to induce STAT6 degradation. However, treatment of IL-4-stimulated cells with calcium ionophores resulted in the absence of phosphorylated STAT6. This effect correlated with the loss of STAT6 protein and was prevented by calpain inhibitors. Cytoplasmic calpains seemed to be responsible for STAT6 degradation. Calpains can target signaling proteins; in this study we found that they can negatively regulate activated STAT6.