Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here, we show that Homo sapiens (Hs) NUF2 is required for stable kinetochore localization of centromere-associated protein E (CENP-E) in HeLa cells. HsNUF2 specifies the kinetochore association of CENP-E by interacting with its C-terminal domain. The region of HsNUF2 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pulldown and yeast two-hybrid assays. Suppression of synthesis of HsNUF2 by small interfering RNA abrogated the localization of CENP-E to the kinetochore, demonstrating the requirement of HsNUF2 for CENP-E kinetochore localization. In addition, depletion of HsNUF2 caused aberrant chromosome segregation. These HsNUF2-suppressed cells displayed reduced tension at kinetochores of bi-orientated chromosomes. Double knockdown of CENP-E and HsNUF2 further abolished the tension at the kinetochores. Our results indicate that HsNUF2 and CENP-E are required for organization of stable microtubule-kinetochore attachment that is essential for faithful chromosome segregation in mitosis.