Emerging evidence suggests that DEAH-box polypeptide 32 (DHX32) serves an important role in the progression and metastasis of cancer. However, the role of DHX32 in breast cancer remains to be completely elucidated. The aim of the present study was to evaluate the expression and clinical significance of DHX32 in breast cancer. The reverse transcription-quantitative polymerase chain reaction was performed to analyze DHX32 messenger (m)RNA expression, and western blotting and immunohistochemistry were performed to examine DHX32 protein expression in breast cancer and adjacent non-cancerous tissues. The association in breast cancer between DHX32 expression, clinicopathological features and prognosis was analyzed using 193 breast cancer tissue samples. The results of the present study demonstrated that breast cancer tissues exhibited increased DHX32 mRNA and protein expression compared with adjacent non-cancerous tissues (P<0.001). In addition, DHX32 expression was significantly associated with breast cancer clinical stage (P=0.006), histological grade (P=0.029), lymph node metastasis (P<0.001) and expression of the proliferation marker Ki-67 (P=0.004). Kaplan-Meier estimator analysis indicated that increased DHX32 expression is associated with poor prognosis in patients with breast cancer. Furthermore, the Cox proportional hazards model indicated that DHX32 expression is an independent prognostic factor for decreased overall survival and disease-free survival in patients with breast cancer. In conclusion, the results of the present study suggest that DHX32 overexpression is an unfavorable prognostic biomarker in breast cancer and a potential therapeutic target of future breast cancer treatments.