The transport of cellular cargos is essential for membrane dynamics, chromosome segregation, patterning the cytoplasm to generate macromolecular gradients, and cell-scale morphogenesis. In remarkable similarity to how cargos are moved around within cities, nature utilizes what is called molecular motor proteins to shuttle cargos along the cytoskeleton, a network of tracks/highways inside cells. All these molecular motors are a two-legged nanomachine. In the first part of my talk, I will present our recent study to understand how cytoplasmic dynein, a molecular motor that moves cargos on tracks called microtubules, achieves processive motion. By directly visualizing the movements of dynein’s two motor domains (legs) using single-molecule, high-precision fluorescence microscopy, we found that that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeleton-based motors. In the second part of my talk, I will discuss the different research projects in my lab that aim to understand the mechanism and regulation of molecular motors by developing and applying powerful molecular and biophysical methods including DNA nanotechnology, high-precision single-molecule imaging, and light-based control of single molecules.