Background: Mounting evidences indicate that circular RNAs (circRNAs) are a novel class of noncoding RNAs and play vital roles in the tumorigenesis and aggressiveness including gastric cancer (GC). Nevertheless, the precise functions and underlying mechanisms of circRNAs in GC remain largely unknown. Methods: The Gene Expression Omnibus (GEO) data set GSE163416 was analyzed to screen the key circRNAs in GC. hsa_circ_0006646 was chosen for further study. GC tissues and matched adjacent normal gastric mucosal epithelial tissues were obtained from the Fourth Hospital of Hebei Medical University. The expressions of hsa_circ_0006646 was detected using quantitative real-time polymerase chain reaction (qRT-PCR). hsa_circ_0006646 was knocked down to identify its effects on GC cells. Bioinformatics algorithms were analyzed to predict the microRNA (miRNAs) potentially sponged by hsa_circ_0006646 and its target genes. Fluorescence in situ hybridization (FISH) was conducted to determine the subcellular location of hsa_circ_0006646 and the predicted miRNA. Then, qRT-PCR, luciferase reporter assay, radioimmunoprecipitation assay, Western blotting, and miRNA rescue experiments were used to confirm the hsa_circ_0006646-related regulatory axis in GC. Cell Counting Kit-8 (CCK-8), colony formation, wound healing, and Transwell experiments were performed to determine the effect of the hsa_circ_0006646-related regulatory axis on GC cells' malignant behaviors in vitro. The xenograft tumor mouse model was established to evaluate the effect of hsa_circ_0006646 in vivo. Results: hsa_circ_0006646 exhibited a high expression in GC tissues as compared to corresponding adjacent normal gastric mucosal epithelial tissues and its high expression was positively correlated with TNM stage, lymph node invasion and poor prognosis (P<0.05). Knockdown of hsa_circ_0006646 suppressed the proliferation, colony formation, migration, and invasion in GC cells (all P<0.05). hsa_circ_0006646 upregulated high mobility group box 1 (HMGB1) by sponging miR-665 in GC cells (P<0.05). The hsa_ circ_0006646-miR-665-HMGB1 axis promoted malignant behaviors and epithelial-mesenchymal transition (EMT) in GC cells by activating the Wnt/beta-catenin pathway (P<0.05). The existence of hsa_circ_0006646-miR-665-HMGB 1 axis was confirmed in GC specimens (P<0.05). Consequently, down-regulated hsa_ circ_0006646 inhibited the progression and EMT of GC cells in vivo (P<0.05). Conclusions: For the first time, we demonstrated that hsa_circ_0006646-miR-665-HMGB1 axis exerted its tumor-promoting effects in GC, which suggested that hsa_circ_0006646 could be potentially targeted for GC treatment.