The FEMALE-SPECIFICALLY EXPRESSED RESPONSE REGULATOR (PdFERR) gene in Populus deltoides, a sex determination gene (an orthologous gene of ARR17 in P. tremula), was found to promote femaleness in heterologous expression lines of Arabidopsis. None of the genes in the Arabidopsis genome seem to be orthologous to PdFERR. Although originating from two evolutionarily distant plants, the dioecious poplar FERR might promote femaleness in the hermaphroditic Arabidopsis through an evolutionary consistent regulatory pathway. However, there is no molecular evidence to support this viewpoint. In this study, to identify the shared downstream orthologous gene of PdFERR, we used yeast two-hybrid (Y2H) to screen potential interactors of PdFERR in Arabidopsis. We identified the ETHYLENE RESPONSE FACTOR 96 (AtERF96) and confirmed the interaction via in vivo and in vitro assays. The ERF96 orthologous gene in P. deltoides was also experimentally confirmed to interact with PdFERR. PdFERR could then promote femaleness in poplar or Arabidopsis through interactions with ERF96, which provide a new perspective for understanding the PdFERR gene regulating sex differentiation. This article is protected by copyright. All rights reserved.