Chemically Modified Tetracyclines (CMTs) are antiproteolytic agents that have been shown to inhibit tumor invasiveness and metastasis. CMT 300 has shown promise in phase I clinical trials in patients with Kaposi's Sarcoma, which is characterized by over-production of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF). In this study, we report a novel activity of CMT 308, a 9-amino derivative of CMT 300, on reducing levels of VEGF secreted by breast cancer cell lines. CMT 308, at sub-cytotoxic concentrations, reduced basal levels of secreted VEGF in the poorly invasive MCF-7 cell line as well as the more aggressively invasive MDA-MB-435s cell line in a dose-dependent manner. In addition, CMT 308 also reduced transforming growth factor β (TGFβ)-induced VEGF secretion in both cell lines. While VEGF could be detected in the conditioned media of untreated MCF-7 cells within 4 h, levels of secreted VEGF in CMT 308-treated cells remained undetectable up to 8 h. CMT 308 diminished secretion of VEGF from MCF-7 cells up to 8 h regardless of previous time in culture. CMT 308 did not reduce the levels of basal VEGF mRNA in either cell line, but did reduce pools of total intracellular VEGF protein. Although TGFβ stimulated an increase in VEGF levels in the conditioned media as well as in the cytoplasm, TGFβ treatment did not increase VEGF mRNA levels. Thus, augmented expression of VEGF protein by breast cancer cell lines in the presence of TGFβ appears to involve upregulation at a step beyond transcription. Moreover, the data strongly indicate that in these breast cancer cell lines, CMT 308 reduces VEGF secretion by targeting some post-transcriptional event. The capacity of CMT 308 to diminish levels of a major pro-angiogenic signal makes the nonantimicrobial tetracycline derivative an attractive candidate for anti-angiogenic therapy in management of breast cancer.