Background: Nonadherence to treatment is a prevalent issue in Internet interventions. Guidance from health care professionals has been found to increase treatment adherence rates in Internet interventions for a range of physical and mental disorders. Evaluating different guidance formats of varying intensity is important, particularly with respect to improvement of effectiveness and cost-effectiveness. Identifying predictors of nonadherence allows for the opportunity to better adapt Internet interventions to the needs of participants especially at risk for discontinuing treatment. Objective: The goal of this study was to investigate the influence of different guidance formats (content-focused guidance, adherence-focused guidance, and administrative guidance) on adherence and to identify predictors of nonadherence in an Internet-based mobile-supported stress management intervention (ie, GET.ON Stress) for employees. Methods: The data from the groups who received the intervention were pooled from three randomized controlled trials (RCTs) that evaluated the efficacy of the same Internet-based mobile-supported stress management intervention (N=395). The RCTs only differed in terms of the guidance format (content-focused guidance vs waitlist control, adherence-focused guidance vs waitlist control, administrative guidance vs waitlist control). Adherence was defined by the number of completed treatment modules (0-7). An ANOVA was performed to compare the adherence rates from the different guidance formats. Multiple hierarchical linear regression analysis was conducted to evaluate predictors of nonadherence, which included gender, age, education, symptom-related factors, and hope for improvement. Results: In all, 70.5% (93/132) of the content-focused guidance sample, 68.9% (91/132) of the adherence-focused guidance sample, and 42.0% (55/131) of the participants in the administrative guidance sample completed all treatment modules. Guidance had a significant effect on treatment adherence (F _2,392=11.64, P<.001; w ²=.05). Participants in the content-focused guidance (mean 5.70, SD 2.32) and adherence-focused guidance samples (mean 5.58, SD 2.33) completed significantly more modules than participants in the administrative guidance sample (mean 4.36, SD 2.78; t ₂₂₃=4.53, P<.001; r=.29). Content-focused guidance was not significantly associated with higher adherence compared to adherence-focused guidance (t ₂₆₂=0.42, P=.67; r=.03). The effect size of r=.03 (95% CI-0.09 to 0.15) did not pass the equivalence margin of r=.20 and the upper bound of the 95% CI lay below the predefined margin, indicating equivalence between adherence-focused guidance and content-focused guidance. Beyond the influence of guidance, none of the predictors significantly predicted nonadherence. Conclusions: Guidance has been shown to be an influential factor in promoting adherence to an Internet-based mobile-supported stress management intervention. Adherence-focused guidance, which included email reminders and feedback on demand, was equivalent to content-focused guidance with regular feedback while requiring only approximately a quarter of the coaching resources. This could be a promising discovery in terms of cost-effectiveness. However, even after considering guidance, sociodemographic, and symptom-related characteristics, most interindividual differences in nonadherence remain unexplained.