The histone deacetylase (HDACs) family contains a family of enzymes, which are involved in modulating a wide range of cellular processes, such as proliferation, differentiation, apoptosis, and cell cycle progression. However, the biological function of HDAC5 in colorectal cancer has not been well established. In the current research, our data showed that the mRNA and protein levels of HDAC5 were up-regulated in human colorectal cancer cell lines. CCK-8 assay showed that overexpression of HDAC5 significantly promoted the proliferation of colorectal cancer cell lines including SW480 and HCT116. On the contrary, HDAC5 knockdown using small interfering RNA suppressed cell growth in colorectal tumor cells. At the molecular level, we demonstrated that HDAC5 promoted the expression of DLL4. In addition, down-regulation of DLL4 diminished the proliferative effects of HDAC5 in human colorectal cancer cells. Taken together, these results suggest that HDAC5 elevates the proliferation of colorectal cancer cells through up-regulation of DLL4. The current study might provide novel potential therapeutic targets in the treatment of colorectal cancer.