In gastric cancer, >15% of cases are associated with the amplification of human epidermal growth factor receptor 2 (HER2), which leads to poor clinical outcomes. Lapatinib, a potent ATP‑competitive inhibitor, is a small, orally active molecule, which inhibits the tyrosine kinases of HER2 and epidermal growth factor receptor type 1. The activation of receptor tyrosine kinases can contribute to lapatinib resistance in HER2‑positive gastric cancer. The aim of the present study was to explore the effects of miR‑494 and FGFR2 in regulation of cancer‑initiating cell phenotypes and therapeutic efficiency of lapatinib in HER2‑positive gastric cancer. Western blot analysis was used to identify that the expression of fibroblast growth factor receptor 2 (FGFR2), a receptor tyrosine kinase, was upregulated in gastric cancer tissues. Formation of cancer initiating cells (CICs) and resistance to lapatinib were determined using sphere growth assay and MTT assay, respectively. The overexpression of FGFR2 promoted the generation of cancer‑initiating cells (CICs) and resistance to lapatinib in HER2‑positive gastric cancer YCC1 cells. In addition, it was observed that overexpression of microRNA (miR)‑494 downregulated the protein expression of FGFR2, inhibited the formation of CICs and reversed lapatinib resistance in YCC1‑F cells (HER2‑positive, FGFR2 overexpressing and lapatinib‑resistant gastric cancer cells). Therefore, it was concluded that miR‑494 inhibited the CIC phenotype and reversed resistance to lapatinib by inhibiting FGFR2 in HER2‑positive gastric cancer.