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Tomato fruit colors are directly associated with their appearance quality and nutritional value. However, tomato fruit color formation is an intricate biological process that remains elusive. In this work we characterized a tomato yellow fruited tomato 3(yft3, e9292, Solanum lycopersicum) mutant with yellow fruits. By the map-based cloning approach, we identified a transversion mutation (A2117C) in the YFT3gene encoding a putative isopentenyl diphosphate isomerase (SlIDI1) enzyme, which may function in the isoprenoid biosynthetic pathway by catalyzing conversion between isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The mutated YFT3(A2117C) (designated YFT3 allele) and the YFT3genes did not show expression difference at protein level, and their encoded YFT3 allelic (S126R) and YFT3 proteins were both localized in plastids. However, the transcript levels of eight genes (DXR, DXS, HDR, PSY1, CRTISO, CYCB, CYP97A, and NCED) associated with carotenoid synthesis were upregulated in fruits of both yft3and YFT3knockout (YFT3-KO) lines at 35 and 47 days post-anthesis compared with the red-fruit tomato cultivar (M82). In vitroand in vivobiochemical analyses indicated that YFT3 (S126R) possessed much lower enzymatic activities than the YFT3 protein, indicating that the S126R mutation can impair YFT3 activity. Molecular docking analysis showed that the YFT3 allele has higher ability to recruit isopentenyl pyrophosphate (IPP), but abolishes attachment of the Mg2+cofactor to IPP, suggesting that Ser126 is a critical residue for YTF3 biochemical and physiological functions. As a result, the yft3mutant tomato line has low carotenoid accumulation and abnormal chromoplast development, which results in yellow ripe fruits. This study provides new insights into molecular mechanisms of tomato fruit color formation and development.