Signal transducer and activator of transcription 3 (STAT3) is a transcription factor involved in multiple fundamental biological processes and a key player in cancer development and progression. STAT3 is activated upon tyrosine phosphorylation and is constitutively active in various malignancies; therefore, the expression of pSTAT3 has been recognized as a predictor of poor survival. STAT3 encodes two alternatively-spliced STAT3 isoforms: the full-length STAT3α isoform and the truncated STAT3β isoform. These isoforms have been suggested as the reason for the occasionally observed opposing roles of STAT3 in cancer: an oncogene, on one hand, and a tumor suppressor on the other. To investigate their roles in aggressive breast cancer, we separately silenced each isoform and found that they affect each other's activation, impacting cell viability, cytokine expression, and migration. Silencing specific isoforms can lead to a more favorable balance of activated STAT3 proteins in the cell. Distinguishing between the two isoforms and their active forms is crucial for STAT3-related cancer diagnosis and therapy.