As a family of post-transcriptional regulator of gene expression, the microRNAs (miRNAs) control a wide array of biological processes including cell differentiation, proliferation and apoptosis, and the dysregulation of miRNAs is a hallmark of cancer. Here, we found that the microRNA-191 (miR-191) was at a high-expression level in human gastric adenocarcinoma cell line MGC803 and human gastric cancer tissues. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays showed that miR-191 could promote cell growth and suppress apoptosis of MGC803 cells. The N-deacetylase/N-sulfotransferase 1 (NDST1) was confirmed to be a direct target gene of miR-191 by enhanced green fluorescent protein reporter experiment. The mRNA and protein levels of NDST1 were inversely correlated with miR-191 in MGC803 cells, suggesting the negative regulation of NDST1 by miR-191. Furthermore, NDST1 played an inhibitory role and could suppress MGC803 cell proliferation. Our findings suggested that miR-191 could act as an oncogene in MGC803 cells, and the cellular function was partially due to its negative regulation of NDST1.