The U.S. Food and Drug Administration (FDA) developed a public education campaign, The Real Cost, that reduced youth susceptibility to tobacco product use. We sought to identify the mechanisms that may underlie the impact of The Real Cost ads on susceptibility to vaping to inform youth tobacco prevention campaigns. Our online randomized controlled trial (clinicaltrials.gov, identifier NCT04836455) examined a large sample of U.S. adolescents (n = 1,348) who had multiple exposures to Real Cost ads or control videos over a 3-week period in 2021. To examine potential mediating pathways between The Real Cost ads and susceptibility to vaping, we examined theory-based psychosocial and message-related variables. The largest impact of The Real Cost ads on susceptibility was via more negative attitudes toward vaping ([beta][subscript a]*[beta][subscript b]= -0.16; 95% confidence interval [CI] = [-0.25, -0.06]). Other mediation paths were via improved health harm risk beliefs ([beta][subscript a]*[beta][subscript b] = -0.08; 95% CI = [-0.13, -0.04]), addiction risk beliefs ([beta][subscript a]*[beta][subscript b] = -0.04; 95% CI = [-0.06, -0.01]), injunctive norms against vaping ([beta][subscript a]*[beta][subscript b] = -0.05; 95% CI = [-0.09, -0.02]), negative affect ([beta][subscript a]*[beta][subscript b] = -0.05; 95% CI = [-0.08, -0.02]), and cognitive elaboration ([beta][subscript a]*[beta][subscript b] = -0.03; 95% CI = [-0.05, -0.003]). Our findings suggest that ads that target negative attitudes may decrease susceptibility to vaping among youth. Our findings also introduce normative pressure as a novel factor that may be important for vaping prevention messages.