Breast cancer, which is the most common type of cancer among women, is a heterogenous disease. It results from progressive accumulation of genetic and epigenetic alterations in different genes. The Dok1 protein has been identified as the major substrate of protein tyrosine kinases in hematopoietic cells. It is considered as a tumor suppressor due to the reports which describe its inhibitory effect on major oncogenic signaling pathways such as Mek/Erk/PI3k/Akt and Wnt/beta-catenin. In this study, we investigated the mutation frequency of the DOK1 gene in 118 breast tumors using Sanger sequencing and DOK1 mRNA expression level in 63 breast cancer samples using qRT-PCR methods. Although the mutation frequency was low DOK1 mRNA expression levels were significantly reduced (63.5%) in the tumors compared to adjacent non-cancerous tissue. We also correlated expression changes with clinicopathological characteristics. Low mRNA levels correlated with age (p = 0.01) and c-erbB-2 (p = 0.05). In most of the previous reports, down-regulation of DOK1 mRNA expression has been associated with promoter methylation. We identified four different coding sequence alterations in 5.1% (6/118) of the tumor samples. However, all of these alterations were located in the functional domains of the protein. Therefore, these mutations may affect the function and/or cellular localization of the protein and contribute to cancer progression by this way. In conclusion our data indicate that DOK1 acts as a tumor suppressor in breast cancer and association of Dok1 with the c-erbB-2 mediated mechanism of action in breast cancer needs to be investigated.