Breast cancer is the most frequent malignancy diagnosed in women, and Gemcitabine-based therapy is frequently used to treat late-stage breast cancer. miR-199a-5p plays a tumor-suppressive role in breast cancer. This work aimed to explore the mechanism of miR-199a-5p plus Gemcitabine in breast cancer cells. Expression of miR-199a-5p was measured by RT-qPCR, while expression of vascular endothelial growth factor A (VEGFA) was measured by Western blot and RT-qPCR. Overexpression of miR-199a-5p and/or silencing of VEGFA was obtained using transfection in breast cancer cells (MCF-7 and MDA-MB-231). Functional experiments were performed to explore cell viability, apoptosis rate, and expressions of apoptosis-related genes: cell viability was assessed by MTT staining, apoptosis rate was recorded by flow cytometry, and Western blot was used to evaluate the expressions of Bcl-2, Bax and cleaved caspase 3. The signaling pathway was studied with respect to AKT activity via determination of p-AKT expression levels. Our study found that miR-199a-5p was downregulated and VEGFA was upregulated in breast cancer tissues and cells. Overexpression of miR-199a-5p and/or silencing of VEGFA contributed to cell apoptosis and inhibited cell viability, which was promoted by Gemcitabine. VEGFA was a downstream target of miR-199a-5p, and was negatively regulated by Gemcitabine. Moreover, Gemcitabine aggravated the miR-199a-5p-induced suppression of the VEGFA level and AKT activity in breast cancer cells. Our data show that Gemcitabine aggravates miR-199a-5p-mediated VEGFA downregulation and apoptosis via inactivating the AKT signaling pathway in breast cancer cells, indicating a novel promising combined therapy of miR-199a-5p overexpression and Gemcitabine.