ABSTRACT: Purpose: Plant trichomes are hair-like protuberances developing from epidermal cells of most terrestrial plants, serve as an excellent system for analyzing the molecular mechanisms underlying cell fate choices, cell cycle control, and cell morphogenesis. Regulation of trichomes in arabidopsis has been well characterized. However, the mechanisms of multicellular trichomes is unclear. To further identify the genes responsible for the SlCycB2-mediated phenotype change, we compared the gene expression profiles of SlCycB2-OE and RNAi plants by RNA-seq. Methods: The transcriptomes of between SlCycB2-OE and RNAi in tomato (Solanum lycopersicum) cultivar Ailsa Craig (AC) by RNA-seq analysis were evaluated using the Illumina HiSeq™ 2500 sequencing platform. Raw sequences were filtered and the resulting sets of clean reads were used for the following analysis by Hisat2 and DEGseq software. qRT-PCR validation was performed using SYBR Green assays. Results: In this study, we determined that SlCycB2 encodes a nuclear localized protein, which is highly expressed in trichomes, stigmas, hypocotyls and stems, and induced by the light, suppressed under the dark. Transgene analysis showed that suppression of SlCycB2 could promote type III and type V trichomes formation. On the contrary, overexpression of SlCycB2 could result in nearly disappearance of all non-glandular trichomes (including type III and V), as well as the glandular ones of type I and type VI. Detection of secondary metabolites indicated that the production of monoterpene and sesquiterpene were significantly decreased in SlCycB2-OE plants, which thus caused the reduction of the defense against Prodenia litura. Transcriptome profile demonstrated that the differential expressed genes mainly participate in the biosynthesis of terpenes, cutin, suberine and wax. Furthermore, we identified several homologs of this gene in many plants, SlCycB3 in tomato, NtCycB2 in tobacco, AtCycB2 in Arabidopsis, which have similar regulatory functions in trichome formation. SlCycB2 overexpression also led to abnormal flower with the unclosed stamen, shortened style and aberrant pollen. Conclusions: The results demonstrate that SlCycB2 play a critical role in multicellular trichome formation, secondary metabolite biosynthesis, Prodenia litura defense and reproductive organ development in tomato. The similar roles of its homologs in multicellular trichome formation suggest that Solanaceous species may share common regulatory pathway.