Updated project metadata. Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Currently available inhibitors target Smoothened, which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smoothened are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2 by using achemical proteomics approach. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and a pseudoprometaphase delay. Dynarrestin reversibly inhibits intraflagellar transport (IFT) of the cargo IFT88 and flux of Smoothened within cilia without interfering with ciliogenesis as well as suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable new tool for probing cytoplasmic dynein-dependent cellular processes and a promising lead compound for medicinal chemistry programs aimed at development of anti-cancer drugs.