In this study, HER2 RNA aptamers were conjugated to mertansine (DM1) and the anti-cancer effectiveness of the conjugate was evaluated in HER2-overexpressing breast cancer models. The conjugate of HER2 aptamer and anticancer drug DM1 (aptamer-drug conjugate, ApDC) was prepared and analyzed using HPLC and mass spectrometry. The cell-binding affinity and cytotoxicity of the conjugate were determined using confocal microscopy and WST-1 assay. The in vivo anti-tumoral efficacy of ApDC was also evaluated in mice carrying BT-474 breast tumors overexpressing HER2. The synthesized HER2-specific RNA aptamers were able to specifically and efficiently bind to HER-positive BT-474 breast cancer cells, but not to HER2-negative MDA-MB-231 breast cancer cells. Also, the HER2-specific ApDC showed strong toxicity to the target cells, BT-474, but not to MDA-MB-231 cells. According to the in vivo analyses drawn from the mouse xenografts of BT-747 tumor, the ApDC was able to more effectively inhibit the tumor growth. Compared to the control group, the mice treated with the ApDC showed a significant reduction of tumor growth. Besides, any significant body weight losses or hepatic toxicities were monitored in the ApDC-treated mice. This research suggests the HER2 aptamer-DM1 conjugate as a target-specific anti-cancer modality and provides experimental evidence supporting its enhanced effectiveness for HER2-overexpressing target tumors. This type of aptamer-conjugated anticancer drug would be utilized as a platform structure for the development of versatile targeted high-performance anticancer drugs by adopting the easy deformability and high affinity of aptamers.