Nitric oxide (NO) is a gaseous regulatory factor produced by NO synthases (NOS) and it plays several critical roles via S-nitrosylation of protein cysteine residues. Histone deacetylase (HDAC) functions in the maintenance/balance of chromatin acetylation and contributes to transcriptional supression. It has been reported that S-nitrosylation of HDAC2 is involved in the regulation of deacetylase activity. However, it remains unknown whether other subtypes of the HDAC family are S-nitrosylated. In the present study, we found that HDAC6 is a target of NO. A biotin-switch assay revealed that endogenous HDAC6 is S-nitrosylated by both NO donors and NO derived from the inducible type of NOS in cells treated with cytokines. NO led to suppressed deacetylase activity in vitro and increased acetylated α-tubulin, a major substrate for HDAC6, in A549 cells. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.