Gastric cancer (GC) ranks among the top five malignant tumors worldwide by the incidence and mortality rate. However, the mechanisms underlying its progression are poorly understood. In this study, we investigated the role of SIRT1, a class III deacetylase, in the invasion and metastasis of GC. Here, we found that knockdown of SIRT1 promoted GC cell migration and invasion in vitro and metastasis in vivo. Forced expression of SIRT1 in GC cells had the opposite effects. Then, we used mRNA microarray to identify the target genes that are regulated by SIRT1 and found that ARHGAP5 was downregulated by SIRT1. The results of the mRNA microarray were confirmed in several GC cell lines. Furthermore, SIRT1 inhibited the expression of ARHGAP5 by physically associating with transcription factor c-JUN and deacetylating and inhibiting the transcriptional activity of c-JUN. Then the expression dynamics and clinical significance of ARHGAP5 were analyzed using clinical samples and database. The expression of ARHGAP5 was increased in GC, and positively correlated with tumor size, tumor infiltration, lymph node metastasis, and clinical stage. And multivariate analyses indicated that ARHGAP5 served as an independent prognostic marker of GC. In addition, the biological effects of ARHGAP5 in SIRT1-mediated inhibition of GC migration and invasion were investigated using both in vitro and in vivo models. Silencing of ARHGAP5 considerably inhibited the migration and invasion of GC, and ARHGAP5 was found to be involved in the SIRT1-mediated inhibition of GC migration and invasion. Our results indicate that SIRT1 suppresses migration and invasion of GC by downregulating ARHGAP5 through an interaction with c-JUN, and these phenomena represent a novel mechanism of the antitumor action of SIRT1.