A novel series of histone deacetylase inhibitors combining N-hydroxycinnamamide bioactive fragment and indole bioactive fragment was designed and synthesized. Several compounds (17c, 17g, 17h, 17j and 17k) exhibited comparable even superior total HDACs inhibitory activity and in vitro antiproliferative activities relative to the approved drug SAHA. A representative compound 17a with moderate HDACs inhibition was progressed to isoform selectivity profile, western blot analysis and in vivo antitumor assay. Although HDACs isoform selectivity of 17a was similar to that of SAHA, our western blot results indicated that intracellular effects of 17a at 1 μM were class I selective. It was noteworthy that the effect on histone H4 acetylation of SAHA decreased with time while the effect on histone H4 acetylation of 17a maintained even increased. Most importantly, compound 17a exhibited promising in vivo antitumor activity in a U937 xenograft model.