As a scaffold protein, Receptor for Activated C Kinase la (RACK1) interacts with many proteins and is involved in multiple biological processes in Arabidopsis. However, the global RACK1 protein interaction network in higher plants remains poorly understood. Here, we generated a yeast two-hybrid library using mixed samples from different developmental stages of Arabidopsis thaliana. Using RACK1a as bait, we performed a comprehensive screening of the resulting library to identify RACK1a interactors at the whole-transcriptome level. We selected 1065 independent positive clones that led to the identification of 215 RACK1a interactors. We classified these interactors into six groups according to their potential functions. Several interactors were selected for bimolecular fluorescence complementation (BiFC) analysis and their interaction with RACK1a was confirmed in vivo. Our results provide further insight into the molecular mechanisms through which RACK1a regulates various growth and development processes in higher plants. (C) 2019 Published by Elsevier Inc.