Abstract The MAPK pathway mediates TLR signaling during innate immune responses. We discovered previously that MKP-1 is acetylated, enhancing its interaction with its MAPK substrates and deactivating TLR signaling. As HDACs modulate inflammation by deacetylating histone and nonhistone proteins, we hypothesized that HDACs may regulate LPS-induced inflammation by deacetylating MKP-1. We found that mouse macrophages expressed a subset of HDAC isoforms (HDAC1, HDAC2, and HDAC3), which all interacted with MKP-1. Genetic silencing or pharmacologic inhibition of HDAC1, −2, and −3 increased MKP-1 acetylation in cells. Furthermore, knockdown or pharmacologic inhibition of HDAC1, −2, and −3 decreased LPS-induced phosphorylation of the MAPK member p38. Also, pharmacologic inhibition of HDAC did not decrease MAPK signaling in MKP-1 null cells. Finally, inhibition of HDAC1, −2, and −3 decreased LPS-induced expression of TNF-α, IL-1β, iNOS (NOS2), and nitrite synthesis. Taken together, our results show that HDAC1, −2, and −3 deacetylate MKP-1 and that this post-translational modification increases MAPK signaling and innate immune signaling. Thus, HDAC1, −2, and −3 isoforms are potential therapeutic targets in inflammatory diseases.