Introduction <p>OBI-858 is a novel 760&#xa0;kDa botulinum toxin type A complex produced by <i>Clostridium botulinum</i>, and currently under development for esthetic applications. This phase II clinical trial aimed to evaluate the efficacy, safety, and optimal dose of OBI-858 for treating moderate-to-severe glabellar lines.</p> Methods <p>In this multicenter, randomized, double-blind, placebo-controlled study, subjects received a single dose of OBI-858 (10U or 20U) or placebo for the treatment of glabellar lines. Subjects were followed for 24-week with efficacy assessed by Facial Wrinkle Scale (FWS), which grades dynamic glabellar line severity (GLS) and Global Aesthetic Improvement Scale (GAIS), along with subject-reported satisfaction. Safety assessments included treatment-emergent adverse events (AEs) and immunogenicity testing.</p> Results <p>At week 4, 74.42% of subjects in the 20U group and 58.70% in the 10U group achieved ≥ 2-point improvement in Glabellar Line as assessed by both investigators and subjects, compared with 0% in the placebo group (<i>p</i> &lt; 0.001). In the 24-week analysis, both 10U and 20U groups demonstrated a rapid onset of GLS 0 or 1 responder rates by day 3, achieving peak rates by week 2. From week 4 to week 20, the 20U group consistently exhibited superior efficacy, showing higher responder rates. Overall, both groups maintained sustained efficacy through week 24. Furthermore, GAIS and subjects reporting satisfaction scores peaked at week 2 and remained favorable in the 20U group through week 24, with nearly 60% of subjects reporting satisfaction at the study’s end. OBI-858 was well tolerated, with no treatment-related serious AE or new antidrug antibody (ADA) development observed during the study.</p> Conclusions <p>OBI-858 demonstrated a favorable safety profile, with no new ADA development observed during the study, along with dose-dependent efficacy and subject satisfaction in treating glabellar lines.</p> Trial Registration <p>TaiwanClinicalTrials.tw: 1139014205.</p>