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Abstract Background Ascorbic acid (Vitamin C, AsA) is an antioxidant metabolite involved in plant development and environmental stimuli. AsA biosynthesis has been well studied in plants, and MIOX is a critical enzyme in plants AsA biosynthesis pathway. However, Myo-inositol oxygenase (MIOX) gene family members and their involvement in AsA biosynthesis and response to abiotic stress remain unclear. Results In this study, five tomato genes encoding MIOX proteins and possessing MIOX motifs were identified. Structural analysis and distribution mapping showed that 5 MIOX genes contain different intron/exon patterns and unevenly distributed among four chromosomes. Besides, expression analyses indicated the remarkable expression of SlMIOX genes in different plant tissues. Furthermore, transgenic lines were obtained by over-expression of the MIOX4 gene in tomato. The overexpression lines showed a significant increase in total ascorbate in leaves and red fruits compared to control. Expression analysis revealed that increased accumulation of AsA in MIOX4 overexpression lines is possible as a consequence of the multiple genes involved in AsA biosynthesis. Myo inositol (MI) feeding in leaf and fruit implied that the Myo-inositol pathway improved the AsA biosynthesis in leaves and fruits. MIOX4 overexpression lines exhibited a better light response, abiotic stress tolerance, and AsA biosynthesis capacity. Conclusions These results showed that MIOX4 transgenic lines contribute to AsA biosynthesis, evident as better light response and improved oxidative stress tolerance. This study provides the first comprehensive analysis of the MIOX gene family and their involvement in ascorbate biosynthesis in tomato.