K-Ras frequently acquires gain-of-function mutations (K-RasG12D being the most common) that trigger significant transcriptomic and proteomic changes to drive tumorigenesis. Nevertheless, oncogenic K-Ras-induced dysregulation of post-transcriptional regulators such as microRNAs (miRNAs) during oncogenesis is poorly understood. Here, we report that K-RasG12D promotes global suppression of miRNA activity, resulting in the upregulation of hundreds of targets. We constructed a comprehensive profile of physiological miRNA targets in mouse colonic epithelium and tumors expressing K-RasG12D using Halo-enhanced Argonaute pull-down. Combining this with parallel datasets of chromatin accessibility, transcriptome, and proteome, we uncovered that K-RasG12D suppressed the expression of Csnk1a1 and Csnk2a1, subsequently decreasing Ago2 phosphorylation at Ser825/829/832/835. Hypo-phosphorylated Ago2 increased binding to mRNAs while reducing its activity to repress miRNA targets. Our findings connect a potent regulatory mechanism of global miRNA activity to K-Ras in a pathophysiological context and provide a mechanistic link between oncogenic K-Ras and the post-transcriptional upregulation of miRNA targets. Display omitted •Oncogenic K-Ras induces global de-repression of physiological miRNA targets in vivo•K-RasG12D enhances Ago2:mRNA binding, likely through Ago2 hypo-phosphorylation•K-RasG12D transcriptionally suppresses Csnk1a1 and Csnk2a1 expression/activities•Csnk1a1 and Csnk2a1 may hierarchically cooperate in their phosphorylation of Ago2 Shui et al. report the in vivo de-repression of global miRNA targets induced by the oncogenic K-Ras (K-RasG12D) that is paradoxically accompanied by the enhanced Ago2:mRNA binding. Integrating in vivo modeling with omics network analysis, they mechanistically connect the K-Ras oncogene to the dysregulation of hundreds of downstream RNA/proteins.