Developing novel strategies against human colorectal cancer (CRC) cells is needed. Activation of AMP-activated protein kinase (AMPK) could possibly inhibit CRC cells. Protein kinase C ζ (PKCζ) is an AMPK negative regulator. Here we found that PKCζ expression was significantly elevated in human colon cancer tissues and CRC cells. PKCζ upregulation was correlated with AMPK in-activation and mTOR complex 1 (mTORC1) over-activation. Reversely, PKCζ shRNA knockdown activated AMPK signaling and inhibited HT-29 cell proliferation. Significantly, downregulation of microRNA-25-5p (miR-25-5p), a PKCζ-targeting miRNA, could be the cause of PKCζ upregulation. Exogenous expression of miR-25-5p silenced PKCζ to activate AMPK signaling, which inhibited HT-29 cell proliferation. studies showed that HT-29 xenograft growth in mice was inhibited after expressing PKCζ shRNA or miR-25-5p. Collectively, PKCζ could be a novel oncogenic protein of human CRC. PKCζ silence, by targeted-shRNA or miR-25-5p expression, activates AMPK and inhibits HT-29 cell proliferation.