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Fruit quality is an important trait in strawberry and is determined by many factors. The soluble solid content in strawberry fruits is positively related to the phosphorus content. MicroRNA399 (miR399) is involved in the regulation of phosphate (Pi) homeostasis. However, the effect of miR399 on strawberry quality remains unknown. In this study, miR399a-overexpressing transgenic woodland strawberries (Fragaria vesca) were obtained via an Agrobacterium-mediated transformation. The phosphorus (P) content was 1.1-fold to 2.1-fold higher in the leaves and fruits of the miR399a-overexpressing plants than in the wild type (WT). However, the P content in the miR399a-overexpressing plants was decreased by 25% to 45% in the roots. The primary root length of the transgenic lines in both the high-Pi and low-Pi media was shorter than that of the WT. Interestingly, the transgenic lines in pots under Pi-sufficient conditions grew better than the WT, and the fruit quality, including the contents of fructose and glucose and soluble solid, was significantly higher in the transgenic lines than in the WT. The overexpression of miR399a in strawberry can be used to improve the parameters involved in fruit quality and provides information regarding breeding nutrient-improved strawberry.