Glycine residues may play functional and structural roles in membrane proteins. In this work we studied the role of glycine residues in EmrE, a small multidrug transporter from Escherichia coli. EmrE extrudes various drugs across the plasma membrane in exchange with protons and, as a result, confers resistance against their toxic effects. Each of twelve glycine residues was replaced by site-directed mutagenesis. Four out of the twelve glycine residues in EmrE are evolutionary conserved within the SMR family of multidrug transporters. Our analysis reveals that only two (Gly67 and Gly97) out of these four highly conserved residues are essential for the transporter activity. Moreover, two glycine positions that are less conserved, Gly17 and Gly90, demonstrate also a nil phenotype when substituted. Our present results identifying Gly17 and Gly67 as irreplaceable reinforce the importance of previously defined functional clusters. Two essential glycine residues Gly90 and Gly97 form a protein motif in which glycine residues are separated by six other residues (GG7). Upon substitution of glycine in these positions, the protein ability to form dimers is impaired as evaluated by crosslinking and pull-down experiments.