Numerous studies have shown that the multifunctional Homeobox-containing (HOX) D3 gene is involved in various physiological and pathological processes. To elucidate the role and mechanism of HOXD3 in colorectal cancer (CRC), we measured its expression in five CRC cell lines. After determining that HOXD3 was highly expressed in the human RKO cancer cell line, we used lentiviral-mediated small interfering RNAs (siRNAs) to knock down HOXD3 expression and assessed proliferation, cell cycle distribution, apoptosis and colony formation using cell proliferation, flow cytometric, and colony formation assays. The expression of HOXD3 was strongly suppressed in the RKO cells infected with the lentiviruse expressing an HOXD3 short hairpin RNA (shRNA). The downregulation of HOXD3 expression in RKO cells significantly decreased proliferation and colony formation, and increased apoptosis in vitro, compared to the cells infected with the mock control (p<0.01). Moreover, specific downregulation of HOXD3 led to the accumulation of cells at the G2 phase of the cell cycle. Our findings revealed that the HOXD3 gene promotes CRC cell growth and plays a pivotal role in the development and survival of malignant human colorectal cancer cells.