The CDK11 (cyclin-dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110-kDa isoform is expressed at constant levels during the cell cycle and the shorter 58-kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. gamma-Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11(p58) isoform, but not the CDK11(p110) isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11(p58) in centrosome maturation and bipolar spindle morphogenesis.