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Objective. Here, we aim to investigate the microRNA (miR) profiling in human gastric cancer (GC). Methods. Tumoral and matched peritumoral gastric specimens were collected from 12 GC patients who underwent routine surgery. A high-throughput miR sequencing method was applied to detect the aberrantly expressed miRs in a subset of 6 paired samples. The stem-loop quantitative real-time polymerase chain reaction (qRT-PCR) assay was subsequently performed to confirm the sequencing results in the remaining 6 paired samples. The profiling results were also validated in vitro in three human GC cell lines (BGC-823, MGC-803, and GTL-16) and a normal gastric epithelial cell line (GES-1). Results. The miR sequencing approach detected 5 differentially expressed miRs, hsa-miR-132-3p, hsa-miR-155-5p, hsa-miR-19b-3p, hsa-miR-204-5p, and hsa-miR-30a-3p, which were significantly downmodulated between the tumoral and peritumoral GC tissues. Most of the results were further confirmed by qRT-PCR, while no change was observed for hsa-miR-30a-3p. The in vitro finding also agreed with the results of both miR sequencing and qRT-PCR for hsa-miR-204-5p, hsa-miR-155-5p, and hsa-miR-132-3p. Conclusion. Together, our findings may serve to identify new molecular alterations as well as to enrich the miR profiling in human GC.