Abstract: Cytoplasmic dynein is a minus-end-directed microtubule-based motor that couples ATP hydrolysis to force generation to move diverse cargos in cells. One of dynein’s conserved essential regulators is Lis1, a gene that is mutated in the brain developmental disease lissencephaly. Previous studies have shown that Lis1 regulates dynein by anchoring it to microtubules. Here we made the unexpected discovery that Lis1 can also act in an opposing manner to stimulate dynein to release from microtubules. Our work describes the molecular basis for these two mechanisms of Lis1 regulation. Using a combination of cryo-electron microscopy structural analysis and in vitro single molecule-assays we show that dynein’s nucleotide state dictates which mode of regulation Lis1 uses. Based on these mechanistic insights we propose a new model for the cell biological regulation of dynein by Lis1.