Abstract Tomato fruit colors are directly associated with their appearance quality and nutritional value. However, the tomato fruit color formation is an intricate biological process that remain 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 YFT3 gene encoding a putative isopentenyl diphosphate isomerase (SlIDI1) enzyme which may functions in the isoprenoid biosynthetic pathway by catalyzing conversion between isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The mutated YFT3 (A2117C) (designated to YFT3 allele) and the YFT3 genes did not show expression difference at protein level, and their encoded YFT3 allele (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 up-regulated in fruits of both yft3 and YFT3 knockout (YFT3-KO) lines at 35dpa and 47dpa compared to the red-fruit tomato cultivar (M82). The in vitro and in vivo biochemical analyses indicated YFT3(S126R) possessed much lower enzymatic activities than the YFT3 protein, indicating that S126R mutation can impair YFT3 activity. The molecular docking analysis showed that YFT3 allele has higher ability to recruit IPP, but abolishes attachment of the Mg2+ cofactor with IPP, suggesting that Ser126 is a critical residue for YTF3 biochemical and physiological functions. As a result, the yft3 mutant tomato line has low carotenoid accumulation and abnormal chromoplast development, which results in the yellow ripe fruits. This study provides new insights into molecular mechanisms of tomato fruit color formation and development.