Lysine or histone deacetylases (HDACs) remove acetyl groups from lysine residues of numerous proteins, thereby regulating their function, activity and localization. Putative HDAC6 substrate sites, uncovered in the acetylome of HDAC6 knockdown cells, served as basis for the design of an HDAC6-trapping peptide library containing hydroxamic acids. Most probes enriched HDAC6 stronger from cell lysates than HDAC1. Probe sequences derived from α-tubulin, CRTC3, HSP90 and PPIA were confirmed as HDAC6 substrates and proteomics profiling of the corresponding trapping probes revealed preferential enrichment of HDAC6 together with known and previously unknown binding proteins. These included proteins of the NF-κB signaling complex which were independently confirmed as HDAC6 binders. NF-κB protein p50 was deacetylated by HDAC6 counteracting enhanced transcription of NF-κB target genes induced by p50 acetylation. These findings indicate a potential anti-inflammatory function of HDAC6 in NF-κB signaling.