N-myc Downstream Regulated Gene 1 (NDRG1) is a metastasis suppressor protein which suppresses metastasis without affecting primary tumorigenesis. There have been many reports about the anti-metastatic function of NDRG1 in various cancers. However, the regulatory mechanism of NDRG1 at the protein level has not been studied widely. Here, we found that NDRG1 is posttranslationally modified by Small Ubiquitin-like Modifier (SUMO), preferentially by SUMO-2, and the major SUMO acceptor site of NDRG1 is Lys 14. Using various SUMO-2 modification status mimicking NDRG1 mutants, we characterized the role of SUMO-2 modification on NDRG1. SUMO-2 modification does not affect the subcellular distribution of NDRG1. However, the protein stability of NDRG1 is influenced by SUMO-2 modification. We found that both the wildtype and the SUMO modification site mutant form of the NDRG1 protein were very stable but the protein stability of SUMO-2 fused NDRG1 K14R had dramatically decreased. In addition, the expression of p21 is downregulated by overexpression of SUMO-2 fused NDRG1 K14R mutants. These results indicate that SUMO-2 modification is implicated in the modulation of NDRG1 protein level and function. This novel link between SUMO modification and regulation of NDRG1 could be a therapeutic target for treatment of various metastatic cancers. (C) 2015 Elsevier Inc. All rights reserved.