microRNAs (miRNAs) are endogenous 19-25 nucleotide noncoding single-stranded RNAs that regulate gene expression by blocking the translation or decreasing the stability of mRNAs. In this study, we showed that miR-200a expression levels were decreased while mitochondrial transcription factor A (TFAM) mRNA expression levels were increased in breast cancer (BC) tissues and cell lines, and identified TFAM as a novel direct target of miR-200a. Overexpression of miR-200a suppressed TFAM protein expression, mtDNA copy number, and attenuated cell proliferation. Forced expression of TFAM can partly rescue the inhibitory effect of miR-200a in the cells. Taken together, these findings will shed light on the role and mechanism of miR-200a in regulating BC cells growth and mtDNA copy number via miR-200a/TFAM axis, and miR-200a may serve as a potential therapeutic target in BC in the future.