In breast cancer, there has been evidence of atypical activation of signal transduction and activators of transcription 3 (STAT3). Thymoquinone (TQ) exerts its anti‐neoplastic effect through diverse mechanisms, including STAT3 inhibition. The tumor suppressor, microRNA‐125a‐5p was reported to be downregulated in various breast cancer cells. Therefore, we investigated the influence of TQ and/or doxorubicin on microRNA‐125a‐5p and its correlation with STAT3 activation as well as tumor growth in mice bearing solid Ehrlich tumors. We found that TQ markedly suppressed inducible and constitutive phosphorylation of STAT3 in tumor tissue without affecting STAT5. Moreover, it attenuated tumor growth, downregulated STAT3 downstream target proteins, and increased the apoptotic activities of caspase‐3 and ‐9. Interestingly, TQ‐elicited synergism of doxorubicin anti‐neoplastic activity was coupled with upregulation of tumoral microRNA‐125a‐5p. Taken together, the current findings raise the potential of TQ as a promising chemomodulatory adjuvant to augment mammary carcinoma sensitivity to doxorubicin.،