The wo protein and its downstream gene, SlCycB2 have been demonstrated to regulate the trichome development in tomato. It was shown that only gain-of-function mutant form of wo, Wo ^v (wo woolly motif mutant allele) could induce the increase of trichome density. However, it is still unclear the relationships between wo, Wo ^v and SlCycB2 in trichome regulation. In this study, we demonstrated Nbwo (NbWo ^v ) directly regulated the expressions NbCycB2 by binding to the promoter of NbCycB2 and its genomic sequences. As a feedback regulation, NbCycB2 negatively regulates the trichome formation by repressing Nbwo activity at protein level. We further found that the mutations of Nbwo woolly motif could prevent repression of NbWo ^v by NbCycB2, which results in the significant increase of active Nbwo proteins, trichome density and branches. Our results revealed a novel reciprocal mechanism between NbCycB2 and Nbwo during the trichome formation in Nicotiana benthamiana . Highlight: NbCycB2 is specifically expressed in trichomes of Nicotiana benthamiana and represses the Nbwo activity via a negative feedback loop in tobacco trichome developmemt.