AbstractCircRNAs, a class of widespread circular RNAs produced from precursor mRNA back-splicing, have been implicated in regulation of gene expression in eukaryotes, but their biological functions in plants have not yet been elucidated. By deep sequencing of rRNA-removed and RNase R-digested RNA samples we have identified several thousands of putative back-splicing sites in tomato fruit (Solanum lycopersicum) and show that the abundance of some of these circRNAs derived from fruit pigment biosynthesis genes are regulated by fruit ripening. Herein, we overexpressed a circRNA derived from Phytoene Synthase 1 (PSY1) in tomato ‘Ailsa Craig’ and microTom. The PSY1 mRNA abundance, the lycopene and β-carotene accumulation were decreased significantly in the transgenic tomato fruits, likely due to the continuous highly expressed circRNAs and/or the low abundant linear RNAs generated from the overexpression vector. Besides, overexpression of a circRNA derived from Phytoene Desaturase (PDS) showed similar results. Our results provide biological insights into plant circRNAs.