Long non‐coding RNA MIF‐AS1 (lncMIF‐AS1) has been found to be upregulated in the tumor tissues of gastric cancer; however, its importance for the progression of gastric cancer remains unknown. Thus, the present study was designed to determine the role of the lncMIF‐AS1‐based signal transduction pathway in mediating the proliferation and apoptosis of gastric cancer cells. Differentially expressed lncRNAs and mRNAs were screened out using microarray analysis, based on the published data (GSE63288), and validated using quantitative RT‐PCR. Target relationships between lncRNA‐micro RNA (miRNA) and miRNA‐mRNA were predicted by bioinformatics analysis and verified by dual‐luciferase reporter assay. Protein expression of NDUFA4, COX6C and COX5B was detected by western blot. Cell proliferation, cell cycle and apoptosis were determined using colony formation assay and flow cytometry analysis. Oxidative phosphorylation in gastric cancer cells was assessed by levels of oxygen consumption and ATP synthase activity. Expression of lncMIF‐AS1 and NDUFA4 were upregulated in gastric cancer tissues and cells as compared with non‐cancerous gastric tissues and cells (P < .05). MiR‐212‐5p was identified as the most important miRNA linker between lncMIF‐AS1 and NDUFA4, which was negatively regulated by lncMIF‐AS1 and its depletion is the main cause of NDUFA4 overexpression (P < .01). The upregulated expression of NDUFA4 then greatly promoted the proliferation and decreased the apoptosis of gastric cancer cells through activation of the oxidative phosphorylation pathway. Taken together, the present study implies that inhibition of lncMIF‐AS1/miR‐212‐5p/NDUFA4 signal transduction may provide a promising therapeutic target for the treatment of gastric cancer.