Cytokinesis is a key step in the later stage of cell division to partition cellular contents into daughter cells, which undergoes precise regulation in temporal and spatial. However, the underlying mechanisms are still unclear. Here, we show that Hsp90 cochaperone, NudCL2 (NudC-like protein 2), is required for cytokinesis in mammalian cells. NudCL2 localizes to midbody during cytokinesis. Knockout (KO) of NudCL2 using CRISPR/Cas9-based genome editing causes cytokinesis failure and leads to the accumulation of multinucleated cells. To investigate the potential regulator involved in NudCL2-mediated cytokinesis regulation, we performed isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis in WT and NudCL2 KO cells and found hundreds of differentially expressed proteins (KO/WT fold change > 1.2 or < 0.83, p < 0.05) in NudCL2 KO cells. We found that the protein level of RCC2 (a midbody-associated protein) is obviously decrease in NudCL2 KO cells. Further studies display that knockout of NudCL2 induces an increase in RCC2 protein instability and degradation. Our data show that depletion of RCC2 leads to the similar defects as NudCL2 KO. Ectopic expression of RCC2 effectively rescues the cytokinesis failure caused by the loss of NudCL2. Interestingly, our data reveals that Hsp90 localizes to midbody in cytokinesis, and interacts with NudCL2 and RCC2. Inhibition of Hsp90 ATPase activity also causes the RCC2 instability and cytokinesis failure as NudCL2 KO. Moreover, the abnormal phenotypes induced by NudCL2 KO are able to rescue by the ectopic expression of Hsp90, but not vice versa. Taken together, our data suggest that NudCL2 is required to cytokinesis by stabilizing RCC2 at midbody through the Hsp90 pathway, providing a hitherto unrecognized mechanism crucial for cytokinesis regulation.