MDN1/Rea1, as an AAA-type ATPase, is predicted to be the largest protein involved in pre-ribosome maturation in most organisms. However, its function in plant growth and development is poorly understood. Here, we characterized a novel Arabidopsis mutant, dwarf &short root (dsr) 1, which shows pleiotropic developmental phenotypes, such as slow germination, short root, dwarf shoot, and reduced seed set under normal growth conditions. Using positional cloning, we revealed that the AtMDN1 function is impaired by a 'glutamic acid' to 'lysine' change at position 3838 of the amino acid sequence in dsr1. Multiple sequence alignment analysis revealed that the mutated Glu residue, which located in the linker domain of AtMDN1, is extremely conserved among organisms. AtMDN1 is expressed in various tissues, particularly in the shoot apex and root tip. Moreover, the results of transcript profile analyses showed that the dysfunction of AtMDN1 in dsr1 impairs the expression of genes related to plant growth and development, which is tightly associated with the pleiotropic phenotypes of dsr1. Thus, we concluded that the Glu residue plays a vital role in maintaining AtMDN1 functions, which are essential for plant growth and development.