MUC4 is a large, heavily glycosylated transmembrane mucin, that is implicated in the pathogenesis of various types of cancers. To date, no extensive study has been done to check the expression and functional significance of MUC4 in different types of gastric adenocarcinomas. Here, we report the expression profile of MUC4 in gastric adenocarcinomas and its function in poorly differentiated gastric non-signet ring cell carcinoma (non-SRCC) type cells. Immunohistochemical analysis using tissue microarray (TMA) showed a significant difference in MUC4 expression between normal adjacent (n = 45) and gastric adenocarcinoma (n = 83; P < 0.001). MUC4 expression was not associated with tumour type, stage or with the degree of differentiation. To gain further insight into the significance of MUC4 expression in gastric non-SRCC cells, MUC4 was ectopically expressed in AGS, a poorly differentiated gastric non-signet ring cell line. The MUC4 overexpressing cells (AGS-MUC4) showed a significant increase (P < 0.005) in cell motility and a decrease in cellular aggregation as compared with the vector-transfected cells. Furthermore, in vivo tumorigenicity analysis revealed that animals transplanted with the MUC4 overexpressing cells (AGS-MUC4) had a greater incidence of tumours (83%) in comparison to empty vector control (17%). In addition, the expression of MUC4 resulted in enhanced expression of total cellular ErbB2 and phosphorylated ErbB2. In conclusion, our results showed that MUC4 is overexpressed in gastric adenocarcinoma tissues, and that it has a role in promoting aggressive properties in poorly differentiated gastric non-SRCC cells through the activation of the ErbB2 oncoprotein.