Ascorbic acid (AsA) is a crucial water-soluble antioxidant in strawberry fruit, but limited research is currently available on the identification and functional validation of key genes involved in AsA metabolism in strawberries. This study analyzed the FaMDHAR gene family identification, which includes 168 genes. Most of the products of these genes are predicted to exist in the chloroplast and cytoplasm. The promoter region is rich in cis-acting elements related to plant growth and development, stress and light response. Meanwhile, the key geneFaMDHAR50that positively regulates AsA regeneration was identified through comparative transcriptome analysis of ‘Benihoppe’ strawberry (WT) and its natural mutant (MT) with high AsA content (83 mg/100 g FW). The transient overexpression experiment further showed that overexpression ofFaMDHAR50significantly enhanced the AsA content by 38% in strawberry fruit, with the upregulated expression of structural genes involved in AsA biosynthesis (FaGalURandFaGalLDH) and recycling and degradation (FaAPX,FaAOandFaDHAR) compared with that of the control. Moreover, increased sugar (sucrose, glucose and fructose) contents and decreased firmness and citric acid contents were observed in the overexpressed fruit, which were accompanied by the upregulation ofFaSNS,FaSPS,FaCEL1andFaACL, as well as the downregulation ofFaCS. Additionally, the content of pelargonidin 3-glucoside markedly decreased, while cyanidin chloride increased significantly. In summary,FaMDHAR50is a key positive regulatory gene involved in AsA regeneration in strawberry fruit, which also plays an important role in the formation of fruit flavor, apperance and texture during strawberry fruit ripening.