Micropropagation has been a well-established methodology for in vitro regeneration of grapevine (Vitis vinifera L.), usually through shoot apical meristem culture or adventitious bud formation. However, recent studies have shown that micropropagated plants often show genetic discrepancy from the mother plant, due to high levels of PGRs applied. Here we present an efficient protocol for rapid in vitro regeneration of grapevine cv. Agiorgitiko genotypes through nodal culture. Development of a single main shoot was prominent on agar-solidified basal MS medium without any PGR or supplemented with relatively low levels (e.g., up to 2.5 µM) of benzyladenine (BA). At higher BA levels axillary bud growth and shoot multiplication were enhanced, but hyperhydricity was also apparent. Relatively low concentrations of indole-3-butyric acid (IBA) promoted both the frequency of shoots forming roots and the number of roots per shoot. Although the overall yield is rather lower compared with shoot tip culture approaches, present protocol may serve as an alternative for true-to-type regeneration of selected clones of ‘Agiorgitiko’ and possibly of other grapevine cultivars.