Drug resistance is a major cause of cancer‑associated mortality. Epirubicin‑based chemotherapy initially benefits patients with metastatic or advanced gastric cancer; however, tumor recurrence can occur following several courses of treatment. Mitochondrial ribosomal protein L33 (MRPL33)‑long (L) and MRPL33‑short (S), isoforms of MRPL33 that arise from AS, have been reported to regulate cell growth and apoptosis in cancer; however, few studies have evaluated the roles of MRPL33‑L and MRPL33‑S in gastric cancer. In the present study, MRPL33‑L was demonstrated to be significantly more abundant in gastric tumor tissues than the MRPL33‑S isoform. MRPL33‑S promoted chemosensitivity to epirubicin in gastric cancer as demonstrated by a chemoresponse assay; chemosensitivity was suppressed in response to MRPL33‑L. Gene microarray analysis was performed to investigate the underlying mechanisms. Bioinformatic analysis revealed that overexpression of MRPL33‑L and MRPL33‑S served critical roles in transcription, signal transduction and apoptosis. In particular, the phosphoinositide 3‑kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway was markedly regulated. A total of 36 target genes, including PIK3 regulatory subunit α, AKT2, cAMP response element‑binding protein (CREB) 1, forkhead box 3, glycogen synthase kinase 3β and mammalian target of rapamycin, which are involved in the PI3K/AKT signaling pathway, were selected for further investigation via protein‑protein interaction network and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Furthermore, western blot analysis indicated that MRPL33‑S promoted the chemoresponse to epirubicin by deactivating PI3K/AKT/CREB signaling and inducing apoptosis, while MRPL33‑L had the opposite effects. In conclusion, the results of the present study revealed that isoforms S and L of MRPL33, which arise from alternative splicing, exhibited opposing roles in the chemoresponse to epirubicin in gastric cancer via the PI3K/AKT signaling pathway. These findings may contribute to the development of potential therapeutic strategies for the resensitization of patients with gastric cancer to epirubicin treatment.