This study aims to explore the detailed signal transduction mechanism of epidermal growth factor receptor (EGFR)/signal transducers and activators of transcription 3 (STAT3) that contributes to the progression of gastric cancer (GC). The STAT3 expression, phosphorylated STAT3 (pSTAT3) expression, and EGFR expression were evaluated by using molecular detection methods of GC tissues, adjacent non-tumor tissues, GC cell lines, and normal gastric cell line. Cetuximab was administered in each cell line to demonstrate the correlations among the above biomarkers. Survival and relationship analyses were adopted to demonstrate the important mechanism of EGFR/STAT3 signaling pathway contributing to the progression of GC. STAT3 expression, pSTAT3 expression, and EGFR expression in GC tissues were significantly higher than those in adjacent non-tumor tissues, respectively. Similarly, we found that STAT3 expression, pSTAT3 expression, and EGFR expression were much higher in GC cell lines than those in GES-1 cell line. With cetuximab administration, both STAT3 expression and pSTAT3 expression in all GC cell lines decreased simultaneously. With Cox proportional hazards model analysis, pSTAT3 expression was identified as the independent predictors of the overall survival of GC patients, as was EGFR expression. Furthermore, we found that there were significant associations between STAT3 expression, pSTAT3 expression, EGFR expression, and lymph node metastasis in GC tissues. The activation of EGFR/STAT3 signaling pathway may contribute to lymph node metastasis, which can promote the progression of GC.