Background: Long non-coding RNS (lncRNA) GATA6-AS regulates endothelial cell growth, which is involved in many types of human diseases. Purpose: Our study was carried out to investigate the involvement of GATA6-AS in gallbladder cancer (GBC). Patients and methods: Sixty-two patients with GBC were recruited in this study. PCR analysis was performed to determine the expression of GATA6-AS, miR-421 and TIMP-2 in tissues or cell lines. Transwell migration and invasion assay was applied. Results: We found that GATA6-AS was downregulated in tumor tissues than in adjacent healthy tissues of GBC patients, and GATA6-AS expression levels in tumor tissues decreased with the increase of clinical stages. MiR-421 was upregulated in tumor tissues than in adjacent healthy tissues of GBC patients and was inversely correlated with the expression levels of GATA6-AS. MiR-421 overexpression failed to significantly affect GATA6-AS in GBC cells, while GATA6-AS overexpression resulted in inhibited miR-421 expression. GATA6-AS overexpression led to decreased migration and invasion rates of GBC cells. MiR-421 overexpression led to increased migration and invasion rates of GBC. Rescue experiments (co-transfection) showed that miR-421 overexpression led to attenuated effects of GATA6-AS overexpression. Conclusion: GATA6-AS may inhibit cancer cell migration and invasion in GBC by downregulating miR-421.