The four conserved guanine-base-binding motifs of Rab GTPase―G1, G3, G4 and G5―are involved in the binding and hydrolysis of GTP. We obtained the full-length coding sequence of the Arabidopsis thaliana RabD2b mutant allele AtRabD2b^[N121I] within the conserved G4 motif by changing asparagine 121 to isoleucine and studied the effects of N121I mutation on the sublocalization and functions of AtRabD2b. The N121I mutation altered the specific localization of AtRabD2b from Golgi stacks to both Golgi and cytoplasm. AtRabD2b could completely complement the functional defect induced by the mutation of Saccharomyces cerevisiae Ypt1, which is homologous to AtRabD2b in yeast. However, At-RabD2b^[N121I] only partly complemented the function of Ypt1. AtRabD2b^[N121I] transgenic A. thaliana showed dwarf, bushy, sterile and necrotic phenotypes, which differed from that of AtRabD2b-overexpressing Arabidopsis with its abnormal main-inflorescence extension phenotype. The N121I mutation alters the subcellular localization of AtRabD2b and affects its normal functions.