ABSTRACT The relevance of Eucalyptus dunnii has been evidenced mainly for its wood quality and cold tolerance among cultivated subtropical eucalypts. However, rooting is a challenge for its propagation, particularly when adult material is involved. This study aimed to assess the mixotrophism on the in vitro elongation and adventitious rooting phases in Eucalyptus dunnii microcutting. The experimental material used was obtained from a ministumps of Eucalyptus dunnii clones. In order to evaluate gas exchange and sucrose supplementation on in vitro elongation and adventitious rooting, the experiment was prepared in a 3×4 factorial arrangements with three forms of sealing (rigid polypropylene caps with no membrane (0/M), with a membrane (1/M), with three membranes (3/M) and four sucrose concentrations (0, 10, 20 and 30 g.L-1). At 30 days in the elongation phase it was evaluated (length, number of shoots per explant, oxidation, bud vigor, pigment content, leaf area and anatomy) and rooting (length, root diameter and rooting). Results show that sucrose should be added in the culture medium for in vitro elongation and can be reduced to concentrations between 10 and 20 g.L-1. In vitro rooting requires the use of 30 g.L-1 of sucrose. The use of flasks with membranes that allow gas exchange is an effective alternative to promote the in vitro elongation and adventitious rooting of Eucalyptus dunnii microcutting.