Significance Kinesins are a superfamily of ATP-dependent motors that are important for a wide variety of microtubule-based functions in eukaryotic cells. Kinesins have evolved to allow variable tuning of their motor properties, but the link between molecular variation and motor function is largely unknown. To understand this link, we have studied an essential mitotic kinesin, kinesin-5, which is the target of anticancer drugs. We used cryo-electron microscopy to visualize directly sequential conformational changes of structural elements during the motor ATPase cycle. We have identified the contribution of kinesin-5–specific variations to motor function indicating that kinesins indeed are precisely tuned according to cellular function. This insight will be important in designing kinesin-specific inhibitors in different disease contexts.