The deregulated cell cycle is a prominent hallmark of oncogenesis. In addition to the genetic alterations in G1 and S phase, emerging data demonstrate that the loss of checkpoint control at the G2/M transition is also closely involved in neoplastic transformation. Among many modulators of the G2/M transition, the defective regulation of cyclin B1, the regulatory subunit of cyclin dependent kinase 1 (Cdk1), and of Polo-like kinase 1 (Plk1) is attracting increasing attention as both of them are overexpressed in a wide spectrum of human tumors. Cyclin B1 and Plk1 serve as negative prognostic markers for patients with various tumors. In this review, we focus on these two molecules and highlight their roles in normal cell cycle, the involvement in oncogenesis and their potential as intervention targets for antitumor therapy.