Proper chromosome segregation during mitosis is one of the most important processes to ensure genome integrity. During this process, the microtubules are captured by a multi-unit complex called kinetochore. The kinetochore is assembled specifically at centromere through recognizing nucleosomes containing the histone H3 variant CENP-A. CENP-N and CENP-C are the only two kinetochore proteins that specifically recognize CENP-A nucleosomes. There are about 1 in 25 nucleosomes that contain CENP-A at the centromere. Therefore, how these two proteins 'ignore' the abundant H3 nucleosomes to interact selectively with a handful of centromeric CENP-A nucleosomes has important implications for genome stability during cell division. To obtain deep insight into the mechanism behind this, I solved the structure of CENP-A nucleosome in complex with CENP-N by single particle cryo electron microscopy (cryo-EM) at 4 A. Through charge and space complementarity, the unique "RG" loop on CENP-A is decoded by CENP-N. CENP-N also engages in extensive interactions with a long segment of the distorted nucleosomal DNA double helix. These interactions were validated in vitro and in vivo.The DNA ends of CENP-A nucleosome which are disordered in the crystal structure are mostly visible in the cryo-EM structure when it is in complex with CENP-N. By micrococcal nuclease digestion assay, the CENP-A nucleosome DNA ends are shown to be less flexible when CENP-N is presented in solution, which is consistent with structural study. Since CENP-N does not interact with DNA ends directly, the less dynamics on the DNA ends indicate a more stable nucleosome. By quantitative electrophoretic mobility shift assay (EMSA) and electron microscopy, the stabilizing effect of CENP-N on CENP-A nucleosome was confirmed in vitro. However, this effect was not significant in vivo , which indicates that the CENP-A nucleosome stability in vivo is determined by multiple factors. Besides the change on DNA ends of CENP-A nucleosome, the orientation of H4 N-terminal tail is altered due to its interaction with CENP-N, with important implications for the multiple biological processes involving the H4 N-terminal tail, especially with respect to the formation of chromatin higher order structure The structural and functional studies in this thesis shed light on how CENP-N ensures that the kinetochore assembles specifically at the centromere.