Increasing evidence supports an important role for the human epidermal growth factor receptor-2 (HER2) gene and mitogen-activated protein kinase (MAPK) signaling pathways in the progression of human cancers by enhancing cancer cell metastasis and proliferation. However, the relationship between HER2 and MAPK signaling pathways in gastric cancer (GC) remains unclear. In the present study, dual in situ hybridization was performed to detect HER2 gene amplification and reverse transcription-quantitative polymerase chain reaction was used to investigate the mRNA expression of members of the MAPK signaling pathway, including rapidly accelerated fibrosarcoma (RAF), extracellular regulated signal-activated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), in 112 primary GC tissue samples. The results revealed that 19/112 (17%) of tissue samples showed positive amplification of HER2, which was correlated with tumor invasion and metastasis. Upregulation of RAF, ERK, p38, and JNK was also observed in samples associated with metastasis. Moreover, the expression levels of RAF and ERK in samples with HER2 gene amplification were significantly increased compared with those without HER2 amplification. However, the expression levels of both p38 and JNK were not significantly correlated with HER2 gene amplification. Our results simultaneously showed the association between HER2 gene amplification and the expression levels of MAPK signaling pathway proteins and clinicopathologic characteristics in GC. These findings provide the basis for investigating the regulation of MAPK signaling pathways by HER2 and potential therapeutic targets for inhibiting metastasis and invasion in GC.