Aberrant expression of microRNA (miRNA) has been reported in various cancers. To clarify the role of miRNA in gastric carcinogenesis, we performed miRNA microarray analysis and investigated expression changes of miRNAs in a 5-aza-2'-deoxycytidine (5-aza-CdR)-treated gastric cancer cell line, KATO-III. On microarray analysis, five miRNAs were found to be upregulated (>3-fold) after 5-aza-CdR treatment compared with untreated cells. Among them, miR-181c and miR-432AS exhibited CpG islands in their upstream sequences on computational analysis, and their upregulation was verified by reverse transcription-polymerase chain reaction analyses. In particular, miR-181c upregulation was found not only in KATO-III but also in two other gastric and one colorectal cancer cell line with 5-aza-CdR treatment. Decreased expression of miR-181c was observed in 9 of 16 primary gastric carcinoma (GC) cases compared with the corresponding non-cancerous stomach tissues. Hypermethylation signals in the upstream region of miR-181c were observed in some cultured and primary GC cells with negative or low miR-181c expression. Transfection of the precursor miR-181c molecule induced decreased growth of two gastric cancer cell lines, KATO-III and MKN45. As for targets of miR-181c, oncogenic NOTCH4 and KRAS were identified by complementary DNA microarray analysis after precursor miR-181c molecule transfection, computational searches of miRNA target databases and reporter assaying using the 3'-untranslated regions of the two genes. These results indicate that miR-181c may be silenced through methylation and play important roles in gastric carcinogenesis through its target genes, such as NOTCH4 and KRAS.