Cytoskeletal motors drive many essential cellular processes. For example, kinesin-1 transports cargo in a step-wise manner along microtubules. To resolve rotations during stepping, we used optical tweezers combined with an optical microprotractor and torsion balance using highly birefringent microspheres to directly and simultaneously measure the translocation, rotation, force, and torque generated by individual kinesin-1 motors. While, at low adenosine 5-triphosphate (ATP) concentrations, motors did not generate torque, we found that motors translocating along microtubules at saturating ATP concentrations rotated unidirectionally, producing significant torque on the probes. Accounting for the rotational work makes kinesin a highly efficient machine. These results imply that the motor’s gait follows a rotary handover-hand mechanism. Our method is generally applicable to study rotational and linear motion of molecular machines, and our findings have implications for kinesin-driven cellular processes. © 2017, National Academy of Sciences. All rights reserved.