Protein sumoylation has emerged as an important regulatory mechanism for the transcriptional machinery. Sumoylation is a highly dynamic process that is regulated in response to cellular stimuli or pathogenic challenges. Altered activity of the small ubiquitin-like modifier (SUMO) conjugation system is associated with human cancers and inflammation. Thus, understanding the regulation of protein sumoylation is important for the design of SUMO-based therapeutic strategies for the treatment of human diseases. Recent studies indicate that the sumoylation system can be regulated through multiple mechanisms, including the regulation of the expression of various components of the sumoylation pathway, and the modulation of the activity of SUMO enzymes. In addition, extracellular stimuli can signal the nucleus to trigger the rapid promoter recruitment of SUMO E3 ligases, resulting in the immediate repression of transcription. Finally, the sumoylation system can also be regulated through crosstalk with other post-translational modifications, including phosphorylation, ubiquitination, and acetylation.