The present study reports the first efficient in vitro regeneration of Ananas erectifolius via indirect organogenesis. Leaf segments (leaf base, middle, and apex) excised from 3- or 5-week-old in vitro plantlets were cultured on 1/4 strength MS medium supplemented with different concentrations and combinations of plant growth regulators. The explants were also exposed to pulse treatment with thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) for different periods of time. The results showed that using juvenile rather than old explants enhanced the frequency of callus induction (35.0% and 16.0%, respectively). Among the explant types tested, only leaf base segments induced calli; the highest frequency occurred via culture treatment containing 4-amino-3,5,6-trichloropicolinic acid (picloram; 48.57%), 2,4-D (40%), or TDZ (35.71%). However, only calli generated in treatments containing TDZ or N6-isopentenyladenine (2ip) were able to develop shoots (maximum 35.71% and 14.28%, respectively). A combination of TDZ and 2,4-D improved callus induction (60.0%) but did not increase shooting response. However, both callusing and shooting response increased when 10 days of pulse treatment with TDZ and 2,4-D was applied (66.83% and 48.7%, respectively). Rooted plantlets that exhibited normal growth and development were acclimatized in a greenhouse and had a survival rate of 95%.