The evolutionary expansion and folding of the mammalian cerebral cortex resulted from amplification of progenitor cells during embryonic development. This process was reversed in the rodent lineage after splitting from primates, leading to smaller and smooth brains. Genetic mechanisms underlying this secondary loss in rodent evolution remain unknown. We show that microRNA miR-3607 is expressed embryonically in the large cortex of primates and ferret, distant from the primate-rodent lineage, but not in mouse. Experimental expression of miR-3607 in embryonic mouse cortex led to increased Wnt/β-catenin signaling, amplification of radial glia cells (RGCs), and expansion of the ventricular zone (VZ), via blocking the β-catenin inhibitor APC (adenomatous polyposis coli). Accordingly, loss of endogenous miR-3607 in ferret reduced RGC proliferation, while overexpression in human cerebral organoids promoted VZ expansion. Our results identify a gene selected for secondary loss during mammalian evolution to limit RGC amplification and, potentially, cortex size in rodents.