The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF.