In vertebrate cells, chromosomes oscillate to align precisely during metaphase. NuSAP, a microtubule-associated protein, plays a critical role in stabilizing spindle microtubules. In this study, we utilize 3D time-lapse live-cell imaging to monitor the role of NuSAP in chromosome oscillation and identify NuSAP as a novel regulator of the chromokinesin, Kid. Depletion of NuSAP significantly suppresses the amplitude and velocity of chromosome oscillation. We analyse the effects of NuSAP and Kid depletion in monopolar and bipolar cells with or without kinetochore microtubule depletion. Twelve postulated conditions are deciphered to reveal the contribution of NuSAP to the polar force generated at kinetochore microtubules and to the regulation of the polar ejection force generated by Kid, thus revealing a pivotal role of NuSAP in chromosome oscillation.