ImportanceUnderstanding whether prevention advertisements reduce susceptibility to vaping is important owing to concerning levels of adolescent vaping.ObjectiveTo examine whether vaping prevention advertisements from the US Food and Drug Administration (FDA) national Real Cost campaign lead to lower susceptibility to vaping among adolescents.Design, Setting, and ParticipantsFor this 3-group randomized clinical trial with parallel assignment, participants were US adolescents aged 13 to 17 years who were susceptible to vaping or current e-cigarette users, recruited from online panels. Adolescents were randomized to 1 of 2 Real Cost vaping prevention trial groups (health harms– or addiction-themed advertisements) or to a control group (investigator-created neutral videos about vaping). Adolescents completed 4 weekly online surveys at visits 1 to 4 over a 3-week period. Data were analyzed from December 1, 2021, to August 25, 2022.InterventionsAdolescents saw 3 randomly ordered 30-second video advertisements online at each of 3 weekly study visits (visits 1, 2, and 3).Main Outcomes and MeasuresThe primary trial outcome was susceptibility to vaping. Surveys also assessed susceptibility to smoking cigarettes to examine any spillover effects of vaping prevention advertisements on smoking outcomes. Both susceptibility measures had 3 items and ranged from 1 (indicating not susceptible) to 4 (indicating highly susceptible). The primary analyses compared Real Cost groups (combined) with the control group, while exploratory analyses compared the Real Cost groups with each other.ResultsParticipants were 1514 adolescents (1140 [75.3%] boys; mean [SD] age, 15.22 [1.18] years), including 504 randomized to the Real Cost health harms group, 506 randomized to the Real Cost addiction group, and 504 randomized to the control group. Adolescents in the Real Cost groups (combined) had lower susceptibility to vaping at visit 4 than those in the control group (b = −0.21; 95% CI, −0.32 to −0.10). The Real Cost groups did not differ from one another on susceptibility to vaping (visit 4: b = −0.05; 95% CI, −0.17 to 0.07). Adolescents in the Real Cost groups (combined) also had lower susceptibility to smoking cigarettes than those in the control group (b = −0.21; 95% CI, −0.32 to −0.10). For both vaping and smoking, Real Cost groups had less positive attitudes (vaping: b = −0.27; 95% CI, −0.40 to −0.14; smoking: b = −0.23; 95% CI, −0.39 to −0.08) compared with the control group.Conclusions and RelevanceThese findings suggest that vaping prevention advertisements from the FDA Real Cost campaign led to lower adolescent susceptibility to vaping and had beneficial spillover effects on cigarette smoking outcomes. Tobacco prevention campaigns can help reduce youth tobacco use.Trial RegistrationClinicalTrials.gov Identifier: NCT04836455