A critical requirement for mitosis is the distribution of genetic material to the two daughter cells. The central player in this process is the macromolecular kinetochore structure, which binds to both chromosomal DNA and spindle microtubule polymers to direct chromosome alignment and segregation. This review will discuss the key kinetochore activities required for mitotic chromosome segregation, including the recognition of a specific site on each chromosome, kinetochore assembly and the formation of kinetochore-microtubule connections, the generation of force to drive chromosome segregation, and the regulation of kinetochore function to ensure that chromosome segregation occurs with high fidelity.