Ring finger protein 2 (RNF2), also known as RING2 or RING1B, displays oncogenic functions in different types of cancers, yet, the function of RNF2 during mitosis has not been evaluated. A yeast two‑hybrid screen was undertaken using a human HeLa cDNA library to explore and identify proteins that interact with RNF2. Several positive clones, including Polo‑like kinase 1 (Plk1), a critical regulator of mitosis, were identified. The interaction between RNF2 and Plk1 was confirmed using a β‑galactosidase and growth test in selective media, in vitro glutathione S‑transferase pull‑down, and in vivo immunoprecipitation assays. Moreover, we confirmed that RNF2 co‑localized with Plk1 at mitotic chromosomes in the prometaphase and metaphase using an immunofluorescence assay. In addition, our results revealed that Plk1 kinase activity was required for ubiquitin‑dependent degradation of RNF2. These findings provide a new clue for understanding the function of RNF2 during mitotic regulation and tumorigenesis.