Micropropagated banana plantlets represent a good planting material to establish new growing areas or replace old plantations. The plantlets are devoid of pathogens but also of beneficial root inhabitants (e.g., endophytes and arbuscular mycorrhizal fungi [AMF]) that may help plants withstand stresses. Mycorrhization of banana is usually conducted at the hardening/post-hardening phases. Only a few studies have reported their application in vitro, and none have investigated the subsequent impact on ex vitro acclimatization. Here, we report for the first time the effects of in vitro mycorrhization of banana plantlets on growth following transfer to soil. Banana plantlets were pre-mycorrhized under autotrophic conditions. During in vitro culture, heavy root colonization of the banana plantlets was observed. At 5 and 7 wk after transfer to a peatsand soil substrate, the root system remained highly colonized. The growth (pseudostem height) and biomass were significantly higher than those of control plants grown on media without AMF. The mean shoot dry weight of the premycorrhized plants at 5 wk after transplanting was 66.7 and 34.6% greater than those of non-mycorrhized plantlets grown on modified Strullu-Romand (MSR) and Murashige and Skoog (MS) media, respectively, and was also greater at week 7 (by 64.7 and 40%, respectively). In vitro mycorrhization under autotrophic culture conditions thus represents a promising tool for the pre-adaptation of micropropagated banana plantlets to ex vitro conditions.