KLF8 (Krüppel-like factor 8) is a member of the Krüppel transcription factor family that binds CACCC elements in DNA and activates or represses their target genes in a context-dependent manner. Here we present sumoylation as a novel mechanism that regulates KLF8 post-translationally. We found that KLF8 can be covalently modified by small ubiqitin-like modifier (SUMO)-1, SUMO-2, and SUMO-3 in vivo. We showed that KLF8 interacted with the PIAS family of SUMO E3 ligases PIAS1, PIASy, and PIASxalpha but not with E2 SUMO-conjugating enzyme Ubc9. Furthermore, we demonstrated that the E2 and E3 ligases enhanced the sumoylation of KLF8. In addition, site-directed mutagenesis identified lysine 67 as the major sumoylation site on KLF8. Lysine 67 to arginine mutation strongly enhanced activity of KLF8 as a repressor or activator to its physiological target promoters and as an inducer of the G(1) cell cycle progression. Taken together, our results demonstrated that sumoylation of KLF8 negatively regulates its transcriptional activity and cellular functions.