Mitosis is an orchestration of dynamic interactions between spindle microtubules and chromosomes, which is mediated by protein structures that include the kinetochores, and other protein complexes present on chromosomes. PinX1 is a potent telomerase inhibitor in interphase; however, its function in mitosis is not well documented. Here we show that PinX1 is essential for faithful chromosome segregation. Deconvolution microscopic analyses show that PinX1 localizes to nucleoli and telomeres in interphase and relocates to the periphery of chromosomes and the outer plate of the kinetochores in mitosis. Our deletion analyses mapped the kinetochore localization domain of PinX1 to the central region and its chromosome periphery localization domain to the C terminus. Interestingly, the kinetochore localization of PinX1 is dependent on Hec1 and CENP-E. Our biochemical characterization revealed that PinX1 is a novel microtubule-binding protein. Our real time imaging analyses show that suppression of PinX1 by small interference RNA abrogates faithful chromosome segregation and results in anaphase chromatid bridges in mitosis and micronuclei in interphase, suggesting an essential role of PinX1 in chromosome stability. Taken together, the results indicate that PinX1 plays an important role in faithful chromosome segregation in mitosis.