Gastric cancer (GC) is a highly malignant solid tumor of the digestive tract, which is associated with a high mortality rate. Long non-coding RNA (lncRNA) nuclear receptor subfamily 2 group F member 1 antisense RNA 1 (NR2F1-AS1) has been reported to exert a tumor-promoting effect in some types of cancer. The present study aimed to investigate the role of NR2F1-AS1 in GC. The expression levels of NR2F1-AS1 and its potential target gene were measured in GC cell lines. Bioinformatics analysis, an RNA immunoprecipitation assay and a chromatin immunoprecipitation assay were used to determine the binding relationship between NR2F1-AS1 and downstream genes. The effect of NR2F1-AS1 regulatory axis on AGC cell viability, proliferation, migration, invasion and epithelial-mesenchymal transition was evaluated. The results of the present study revealed that the knockdown of NR2F1-AS1 inhibited the proliferation, invasion and migration of GC cells. NR2F1-AS1 also upregulated the expression levels of ST8SIA1 by recruiting transcriptional factor SPI1. Thus, the effects of the knockdown of NR2F1-AS1 on GC cell functions were suggested to occur via regulation of ST8SIA1. In conclusion, the findings of the current study indicated that NR2F1-AS1 may promote the proliferation, invasion and migration of GC cells by recruiting SPI1, to upregulate ST8SIA1 expression. Thus, the regulation of their expression levels may provide a novel direction for the treatment of GC.