Accumulating evidence implicates the transcription factor NF-κB as a positive mediator of cell growth, but the molecular mechanism (s) involved in this process remains largely unknown. Here we use both a skeletal muscle differentiation model and normal diploid fibroblasts to gain insight into how NF-κB regulates cell growth and differentiation. Results obtained with the C2C12 myoblast cell line demonstrate that NF-κB functions as an inhibitor of myogenic differentiation. Myoblasts generated to lack NF-κB activity displayed defects in cellular proliferation and cell cycle exit upon differentiation. An analysis of cell cycle markers revealed that NF-κB activates cyclin D1 expression, and the results showed that this regulatory pathway is one mechanism by which NF-κB inhibits myogenesis. NF-κB regulation of cyclin D1 occurs at the transcriptional level and is mediated by direct binding of NF-κB to multiple sites in the cyclin D1 promoter. Using diploid fibroblasts, we demonstrate that NF-κB is required to induce cyclin D1 expression and pRb hyperphosphorylation and promote G 1-to-S progression. Consistent with results obtained with the C2C12 differentiation model, we show that NF-κB also promotes cell growth in embryonic fibroblasts, correlating with its regulation of cyclin D1. These data therefore identify cyclin D1 as an important transcriptional target of NF-κB and reveal a mechanism to explain how NF-κB is involved in the early phases of the cell cycle to regulate cell growth and differentiation.