Previously, we have reported that gain at chromosome 20q13 is the most common genomic copy number aberration in gastric cancer (GC) (29/30 cases), and that among the genes located in this region, we have identified DDX27, whose expression level shows the highest correlation with genomic copy number, as a candidate therapeutic target for GC. Here, we analyzed the clinicopathological significance of DDX27 using immunohistochemistry and studied its functions using knockdown assays. We found that DDX27 was frequently upregulated in GC tissues (98 of 140 cases, 70%), and significantly associated with venous invasion and liver metastasis. Furthermore, multivariate analysis of GC patients showed that high expression of DDX27 was independently associated with poorer prognosis. In functional assays, knockdown of DDX27 reduced the ability of GC cells to form colonies both on conventional plates and soft agar, but had little effect on their invasiveness. We also found that knockdown of DDX27 reduced the viability of GC cells through inhibition of cell cycle progression independently of apoptosis. Interestingly, DDX27 depletion induced accumulation of TP53 in a TP53 wild-type cell line, AGS, but not in a TP53-deleted cell line, 44As3, although DDX27 knockdown commonly reduced the viability of both, indicating the TP53-dependent and independent cell cycle control of DDX27. Thus, our results suggest that expression of DDX27 contributes to colony formation by GC cells through cell cycle control and may be a potential therapeutic target for GC patients with chromosome gain at 20q13.