The conserved protein Shugoshin (Sgo) plays a role in the maintenance of centromeric cohesion in mitosis and meiosis. Human Shugoshin (hSgo) was first identified as an overexpressed protein in breast cancers. Here we demonstrate that hSgo mediates kinetochore-driven formation of kinetochore -microtubules (MTs) during bipolar spindle assembly. The regulated overexpression of full-length hSgo, or of truncated proteins containing both the conserved N-terminal coiled-coil domain and C-terminal basic domain, resulted in hSgo localization at centromere at early mitosis and was associated with aberrant nucleation and formation of bundles of kinetochore-MTs. The mid-portion of hSgo, between the N- and C-terminal domains, includes both a functional domain for centromeric cohesion and a regulatory domain for spindle assembly. The cells overexpressing natural alternative splicing isoforms, which are almost completely defective for the mid-portion of the hSgo protein, showed premature centromere separation (PCS) and aberrant MT connections. These isoforms are mildly overexpressed in HEK293 cells. On the other hand, cells expressing a truncated protein, defective in the lysine-rich region of the mid-portion, arrested at mitosis due to persistent abnormal MT connections and not because of PCS. Aberrant MT connections were predominantly observed in spindle regions where chromosomes were clustered. Interestingly, we also found that hSgo is rapidly exchanged at kinetochores at early mitosis. Based on these results, we conclude that hSgo may be diffusible and have a role in proper kinetochores MTs attachment.