Ibervillea sonorae is a medicinal plant mainly used to treat diabetes, ulcers, and other metabolic disorders. A regeneration protocol using internode segments containing axillary buds grown on Gamborg medium (B5) supplemented with 0.5 mg·L−1 α-naphthalene-acetic acid (NAA), 0.5 mg·L−1 N6-benzyladenine (BA), and 1.0 mg·L−1 indole-3-acetic acid (IAA) successfully regenerated shoots in I. sonorae explants. The induction of organogenic calli attained 100% efficiency. The highest percent shoot production was 87.5% with a mean of 9.17 shoots per explant on day 15, and the maximum length of 5.8 cm was observed on day 21. Regenerated shoots induced roots in B5 medium supplemented with 0.5–3.0 mg·L−1 indole-3-butyric acid (IBA). The maximum rooting frequency observed in the medium containing 2.0 mg·L−1 IBA was 83.3% which promoted long, thick roots on day 21. The plantlets with emerging roots grown at the culture facility attained 50% survival after acclimatization for 30 d. The account describes a simple and efficient protocol for in vitro plant regeneration, and this micropropagation procedure offers an alternative for preservation of this medicinal plant.