S6 kinases (S6Ks) are mechanistic target of rapamycin substrates that participate in cell growth control. S6Ks phosphorylate ribosomal protein S6 (rpS6) and additional proteins involved in the translational machinery, although the functional roles of these modifications remain elusive. Here we analyze the S6K-dependent transcriptional and translational regulation of gene expression by comparing whole-genome microarray of total and polysomal mouse liver RNA after feeding. We show that tissue lacking S6Ks 1 and 2 (S6K1 and S6K2), displays a defect in the ribosome biogenesis (RiBi) transcriptional program after feeding. Over 75% of RiBi factors are controlled by S6K, including Nop56, Nop14, Gar1, Rrp9, Rrp15, Rrp12 and Pwp2 nucleolar proteins. Importantly, the reduced activity of RiBi transcriptional promoters in S6K1; S6K2−/− cells is also observed in rpS6 knock-in mutants that cannot be phosphorylated. As ribosomal protein synthesis is not affected by these mutations, our data reveal a distinct and specific aspect of RiBi under the control of rpS6 kinase activity, that is, the RiBi transcriptional program.