DPY30, a core subunit of the SET1/MLL histone H3K4 methyltransferase complexes, plays an important role in diverse biological functions through the epigenetic regulation of gene transcription, especially in cancer development. However, its involvement in human colorectal carcinoma (CRC) has not been elucidated yet. Here we demonstrated that DPY30 was overexpressed in CRC tissues, and significantly associated with pathological grading, tumor size, TNM stage, and tumor location. Furthermore, DPY30 knockdown remarkably suppressed the CRC cell proliferation through downregulation of PCNA and Ki67in vitroandin vivo, simultaneously induced cell cycle arrest at S phase by downregulating Cyclin A2. In the mechanistic study, RNA-Seq analysis revealed that enriched gene ontology of cell proliferation and cell growth was significantly affected. And ChIP result indicated that DPY30 knockdown inhibited H3 lysine 4 trimethylation (H3K4me3) and attenuated interactions between H3K4me3 with PCNA, Ki67 and cyclin A2 respectively, which led to the decrease of H3K4me3 establishment on their promoter regions. Taken together, our results demonstrate overexpression of DPY30 promotes CRC cell proliferation and cell cycle progression by facilitating the transcription of PCNA, Ki67 and cyclin A2 via mediating H3K4me3. It suggests that DPY30 may serve as a potential therapeutic molecular target for CRC.