We examined immunological responses in patients receiving histone deacetylase (HDAC) inhibition (vorinostat) for graft-versus-host disease prophylaxis after allogeneic hematopoietic cell transplant. Vorinostat treatment increased histone acetylation in peripheral blood mononuclear cells (PBMCs) from treated patients, confirming target HDAC inhibition. HDAC inhibition reduced proinflammatory cytokine levels in plasma and from PBMCs and decreased ex vivo responses of PBMCs to proinflammatory TLR-4 stimuli, but did not alter the number or response of conventional T cells to nonspecific stimuli. However, the numbers of regulatory T cells (Tregs) were increased, which revealed greater demethylation of the Foxp3 T regulatory-specific demethylation region. Vorinostat-treated patients showed increased expression of CD45RA and CD31 on Tregs, and these Tregs demonstrated greater suppression on a per cell basis. Consistent with preclinical findings, HDAC inhibition also increased signal transducer and activator of transcription 3 acetylation and induced indoleamine-2,3-dioxygenase. Our data demonstrate that HDAC inhibition reduces inflammatory responses of PBMC but enhances Tregs after allo-HCT.