rsw3 is a temperature-sensitive mutant of Arabidopsis thaliana showing radially swollen roots and a deficiency in cellulose. The rsw3 gene was identified by a map-based strategy, and shows high similarity to the catalytic α-subunits of glucosidase II from mouse, yeast and potato. These enzymes process N-linked glycans in the ER, so that they bind and then release chaperones as part of the quality control pathway, ensuring correct protein folding. Putative β-subunits for the glucosidase II holoenzyme identified in the Arabidopsis and rice genomes share characteristic motifs (including an HDEL ER-retention signal) with β-subunits in mammals and yeast. The genes encoding the putative α- and β-subunits are single copy and, like the rsw3 phenotype, widely expressed. rsw3 reduces cell number more strongly than cell size in stamen filaments and probably stems. Most features of the rsw3 phenotype are shared with other cellulose-deficient mutants, but some – notably, production of multiple rosettes and a lack of secreted seed mucilage – are not and may reflect glucosidase II affecting processes other than cellulose synthesis. The rsw3 root phenotype develops more slowly than the rsw1 and rsw2 phenotypes when seedlings are transferred to the restrictive temperature. This is consistent with rsw3 reducing glycoprotein delivery from the ER to the plasma membrane whereas rsw1 and rsw2 act more rapidly by affecting the properties of already delivered enzymes.