ζ-carotene desaturase (ZDS) is one of the enzymes that catalyzes the conversion of ζ-carotene to lycopene. However, the role of ZDS has been not studied extensively in tomato. In this study, to examine the role of ZDS in tomato, the expression of ZDS was downregulated by RNA interference. The relative mRNA levels of ZDS in RNAi lines were reduced by more than 75% compared to wild-type plants. The genetically modified plants exhibited altered morphological characteristics such as photobleached leaves, increased leaf length, longer petioles, larger leaf surface areas and whitish-green tomato fruits. Chromatographic determination showed a significant increase in phytoene in both leaves and fruits of the RI lines, whereas other types of carotenoids were significantly decreased compared to controls. qPCR analysis revealed significant changes in the expression of PSY, PDS and LCYB. The endogenous hormones and flavonoid levels were altered in leaves and fruits of ZDS-silenced plants. Electron microscopic studies revealed hampered chloroplast differentiation and disrupted thylakoid structure in the ZDS-suppressed lines. GC–MS analysis showed that the level of several organic acids, amino acids and fatty acids were decreased in the RI lines, whereas the sugars and sugar derivatives were slightly increased compared to controls. Thus, these results imply a potential for ZDS in carotenoid enhancement through genetic engineering of the carotenoid pathway. © 2020, Springer Nature B.V.