KIF3b is a protein of the kinesin-2 family which plays an important role in intraflagellar transport. Testis cancer is a common cancer among young men. Its diagnostic rate is increasing and over half of the cases are seminomas. Many aspects of the mechanism and gene expression background of this cancer remain unclear. Using western-blotting and semi-quantitative PCR we found high protein levels of KIF3b enrichment in seminoma tissue despite the mRNA levels remaining equivalent to that of normal testicular tissues. The distribution of KIF3b was mainly in cells with division potential. Wound-healing assays and cell counting kit assays showed that the knockdown of KIF3b significantly suppressed cell migration ability, viability and number in HeLa cells. Immunofluorescence images during the cell cycle revealed that KIF3b tended to gather at the spindles and was enriched at the central spindle. This indicated that KIF3b may also have direct impacts upon spindle formation and cytokinesis. By counting the numbers of nuclei, spindles and cells, we found that the rates of multipolar division and multi-nucleation were raised in KIF3b-knockdown cells. In this way we demonstrate that KIF3b functions importantly in mitosis and may be essential to seminoma cell division and proliferation as well as being necessary for normal cell division.