Abstract Simmondsia chinensis (jojoba) is an important industrial plant of arid regions generally propagated by seeds or cuttings. Seed propagation has the problem that jojoba is a dioecious plant and sex determination can only be done in plants 3–4 years old. Although in vitro and ex vitro culture of jojoba was developed using different methodologies, no comparative studies of rooting-acclimatization systems with and without A. brasilense inoculation were performed. Established in vitro shoots were induced to root with 0, 25, 50 or 75 μM indole-3-butyric acid (IBA) for 0, 4, 6 or 8 days in modified Murashige and Skoog medium and then, transferred to auxin free medium for 50 days. Maximum rooting percentage evaluated by response surface analysis resulted in 67% with 60 μM IBA for 7 days in A. brasilense inoculated treatment compared to 33% using 50 μM IBA for 5 days in the control treatment. In vitro shoots induced with 50 μM IBA for 6 days and ex vitro shoots induced with 29.5 mM IBA for 20 s were transferred to peat-perlite substrate. Nodal segments obtained from field grown plants were used as explant for ex vitro rooting. Shoots of all treatments were acclimatized in a plug tray containing a mix of sterile peat and perlite and exposed to a regime of gradually decreasing humidity. Some shoots of each treatment were inoculated with A. brasilense Az39 (1.5 × 10 8 UFC mL −1 ) at time of transplant and non-inoculated shoots were the controls. In vitro material showed significantly higher (p ≤ 0.05) rooting percentage in both substrate and in vitro (34% and 37%, respectively) respect to ex vitro source material (9%). A. brasilense Az39 inoculation increased from 33% to 410% the rooting percentage in the different rooting systems. Acclimatization survival rate in plantlets from in vitro rooting system was 63% whereas in plantlets from ex vitro rooting system was 95%. Inoculated treatments significantly decreased jojoba mesophyll thickness and increased cell density compared to uninoculated treatments. Shoots rooted ex vitro showed higher percentage of tannin cells than those rooted in vitro . Foliar epidermis, stomatal density and stomatal index significantly increased in jojoba plants rooted in substrate compared to in vitro treatments. Replacing in vitro by ex vitro rhizogenesis allows simultaneouly to perform rooting and acclimatization which reduces costs of micropropagation and adds efficiency to plant production. In addition, the in vitro plant source allows to reduce roguing and seasonal depence. A. brasilense Az39 increased rooting-acclimatization final yields and produced anatomical features similar to ex vitro plants. So, ex vitro rooting using in vitro plant source and A. brasilense inoculation is recommended.