Gastric cancer (GC) is the most frequent leading cause of cancer‐associated mortality worldwide that is linked to poor prognosis due to the lack of appropriate biomarkers. Our aim was to evaluate the MUC5AC and Oct‐4 expression levels in GC and to assess their association with clinical factors. Immunohistochemical analysis (IHC) and qRT‐PCR were performed in GC patients to examine the MUC5AC and Oct‐4 expression levels. The mRNA level of MUC5AC was significantly decreased in tumour tissues compared with non‐cancerous tissues (1.11 ± 0.69 vs 3.7 ± 0.71; P = .024). On the other hand, Oct‐4 mRNA level was upregulated in tumour tissues as compared to normal tissues (2. 86 ± 0.78 vs 0.87 ± 0.54; P = .0015). Decreased expression of MUC5AC was detected in 27 patients (67.5%), while high to moderate expression levels were observed in 13 cases (32.5%), but in normal tissues the expression levels of MUC5AC were increased (P = .001). The decreased expression of MUC5AC was associated with aggressive tumour characteristics, such as TNM stage (P = .023), histologic type (P = .012) and lymph node metastasis (P = .001). High expression of Oct‐4 was detected in 24 tumour tissues (60%), while 16 cases (40%) showed low expression level. Increased Oct‐4 expression was correlated with clinicopathological characteristics such TNM stage (P = .002), histologic type (P = .008) and lymph node metastasis (P = .001). Our results showed that high Oct‐4 expression and the reduction of MUC5AC expression may be involved in the progression and an unfavorable prognosis of GC.