Malic acid is one of the most important organic acids in strawberry fruits, and its content significantly affects the flavor of strawberry fruits. In this study, using Fragaria × ananassa ‘Benohoppe’ as material, we cloned a new MYB transcription factor (TF)FaMYB73 by RTPCR including 756 bp open reading frame, encoding 252 amino acids. It was predicted that the molecular mass of this protein was 27.6 kD, and pI was 6.8. The FaMYB73 protein contained the conserved R2R3MYB domain and microscopy analysis showed that 35SFaMYB73GFP was located exclusively in the nucleus as expected. Quantitative realtime PCR (qRTPCR) analysis demonstrated that the expression of FaMYB73 had tissue specific and stage specific. FaMYB73 was expressed in different tissues and four developmental stages of fruits. The expression level of FaMYB73 was relatively higher in leaves and flowers, the highest at the white fruit stage and decreased with maturity. The malic acid content detected by HPLC reached the highest at the white fruit stage and decreased thereafter, and was positively correlated with the expression of the FaMYB73 gene. The homologous expression in strawberries found that FaMYB73 overexpression significantly promoted the accumulation of malic acid, and silence distinctly reduced the amounts of malic acid. Two transgenic tobacco lines having the malic acid overaccumulation phenotype were obtained and the transformation was confirmed using quantitative realtime PCR (qRTPCR). The results indicated that FaMYB73 was involved in regulating the synthesis of malic acid in strawberry fruits and provided a theoretical foundation for further exploring the mechanism of FaMYB73 regulating malic acid in strawberry fruits.