Members of the C/EBP transcription factor family regulate cell differentiation and multiple other cellular functions. The cellular levels of C/EBPα, γ, δ, ε, and Gadd153/CHOP are regulated in part by proteasome-dependent degradation. In contrast, mechanisms regulating the degradation of C/EBPβ are poorly understood. We tested the hypothesis that the degradation of C/EBPβ is calpain-dependent. Studies were performed in cultured L6 myotubes (a rat skeletal muscle cell line) because we have found previously that C/EBPβ may be involved in the regulation of muscle proteolysis. Treatment of cultured L6 myotubes with the calpain inhibitors calpeptin and Calpain Inhibitor I and II resulted in increased C/EBPβ concentrations but did not influence cellular levels of the other C/EBP transcription factor family members. Transfection of myoblasts with a plasmid expressing the endogenous calpain inhibitor calpastatin resulted in increased cellular levels of C/EBPβ whereas the opposite result was observed in myoblasts overexpressing µ- or m-calpain. Co-immunoprecipitation provided evidence for protein–protein interaction between C/EBPβ and µ- and m-calpain suggesting that C/EBPβ may be a calpain substrate. This notion was supported by experiments in which immunoprecipitated C/EBPβ was incubated with purified µ-calpain in a cell-free system. The increase in C/EBPβ levels caused by inhibition of calpain activity was accompanied by increased C/EBPβ DNA-binding and gene activation. The present results suggest that C/EBPβ is degraded by a calpain-dependent mechanism in skeletal muscle cells and that the role of calpains is specific for C/EBPβ among different members of the C/EBP transcription factor family. J. Cell. Physiol. 208: 386–398, 2006. © 2006 Wiley-Liss, Inc.