The CCAAT box is a DNA element present in the majority of human promoters, bound by the trimeric NF-Y, composed of NF-YA, NF-YB, and NF-YC subunits. We describe and characterize novel isoforms of one of the two histone-like subunits, NF-YC. The locus generates a minimum of four splicing products, mainly located within the Q-rich activation domain. The abundance of each isoform is cell-dependent; only one major NF-YC isoform is present in a given cell type. The 37- and 50-kDa isoforms are mutually exclusive, and preferential pairings with NF-YA isoforms possess different transcriptional activities, with specific combinations being more active on selected promoters. The transcriptional regulation of the NF-YC locus is also complex, and mRNAs arise from the two promoters P1 and P2. Transient transfections, chromatin immunoprecipitations, and reverse transcription-PCRs indicate that P1 has a robust housekeeping activity; P2 possesses a lower basal activity, but it is induced in response to DNA damage in a p53-dependent way. Alternative promoter usage directly affects NF-YC splicing, with the 50-kDa transcript being excluded from P2. Specific functional inactivation of the 37-kDa isoform affects the basal levels of G(1)/S blocking and pro-apoptotic genes but not G(2)/M promoters. In summary, our data highlight an unexpected degree of complexity and regulation of the NF-YC gene, demonstrating the existence of a discrete cohort of NF-Y trimer subtypes resulting from the functional diversification of Q-rich transactivating subunits and a specific role of the 37-kDa isoform in suppression of the DNA damage-response under growing conditions.