Gastric cancer (GC) is a common heterogeneous disease. The critical roles of microRNA‐340 (miR‐340) in the development and progression of GC were emphasized in accumulating studies. This study aims to examine the regulatory mechanism of miR‐340 in GC cellular processes. Initially, microarray technology was used to identify differentially expressed genes and regulatory miRs in GC. After that, the potential role of miR‐340 in GC was determined via ectopic expression, depletion, and reporter assay experiments. Expression of secreted phosphoprotein 1 (SPP1), miR‐340, phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT) pathway, and epithelial–mesenchymal transition (EMT)‐related genes was measured. Moreover, to further explore the function of miR‐340 in vivo and in vitro, proliferation, apoptosis, migration, invasion, and tumorigenic capacity were evaluated. SPP1 was a target gene of miR‐340 which could then mediate the PI3K/AKT signaling pathway by targeting SPP1 in GC. Furthermore, miR‐340 levels were reduced and SPP1 was enriched in GC tissues and cells, with the PI3K/AKT signaling pathway being activated. Inhibitory effects of upregulated miR‐340 on SPP1 and the PI3K/AKT signaling pathway were confirmed in vivo and in vitro. Overexpression of miR‐340 or the silencing of SPP1 inhibited GC cell proliferation, invasion, migration, and EMT process, but promoted apoptosis of GC cells. Typically, targeting of SPP1 by miR‐340 may contribute to the inhibition of proliferation, migration, invasion, and EMT of GC cells via suppression of PI3K/AKT signaling pathway.