Adult neurogenesis occurs throughout life in the mammalian hippocampus and is essential for memory and mood control. There is significant interest in identifying ways to promote neurogenesis and ensure maintenance of these hippocampal functions. Previous work with a synthetic small molecule, isoxazole 9 (Isx-9), highlighted its neuronal-differentiating properties in vitro. However, the ability of Isx-9 to drive neurogenesis in vivo or improve hippocampal function was unknown. Here we show that Isx-9 promotes neurogenesis in vivo, enhancing the proliferation and differentiation of hippocampal subgranular zone (SGZ) neuroblasts, and the dendritic arborization of adult-generated dentate gyrus neurons. Isx-9 also improves hippocampal function, enhancing memory in the Morris water maze. Notably, Isx-9 enhances neurogenesis and memory without detectable increases in cellular or animal activity or vascularization. Molecular exploration of Isx-9-induced regulation of neurogenesis (via FACS and microarray of SGZ stem and progenitor cells) suggested the involvement of the myocyte-enhancer family of proteins (Mef2). Indeed, transgenic-mediated inducible knockout of all brain-enriched Mef2 isoforms (Mef2a/c/d) specifically from neural stem cells and their progeny confirmed Mef2's requirement for Isx-9-induced increase in hippocampal neurogenesis. Thus, Isx-9 enhances hippocampal neurogenesis and memory in vivo, and its effects are reliant on Mef2, revealing a novel cell-intrinsic molecular pathway regulating adult neurogenesis.—Petrik, D., Jiang, Y., Birnbaum, S. G., Powell, C. M., Kim, M.-S., Hsieh, J., Eisch, A. J. Functional and mechanistic exploration of an adult neurogenesis-promoting small molecule.