Objective This randomized controlled trial (RCT) aimed to evaluate the efficacy of an internet-based stress management intervention (iSMI) among employees compared to a 6-month waitlist control group (WLC) with full access to treatment-as-usual. Method A sample of 264 employees with elevated symptoms of perceived stress (Perceived Stress Scale, PSS-10 >=>22) was assigned to either the iSMI or WLC group. The iSMI consisted of seven sessions and one booster session including problem-solving and emotion regulation techniques. Participants received guidance from an e-coach that focused on improving the adherence to the intervention. Self-report data were assessed at baseline, seven weeks, and six months following randomization. The primary outcome was perceived stress (PSS-10). The secondary outcomes included other relevant mental and work-related health outcomes. Results The iSMI participants showed a significantly higher reduction in perceived stress from baseline to seven weeks [d=0.79, 95% confidence interval (95% CI) 0.54-1.04] and the 6-month follow up (d=0.85, 95% CI 0.59-1.10) compared to controls. Significant moderate-to-large effect sizes were also found for depression, anxiety, emotional exhaustion, sleeping problems, worrying, quality of life (mental health), psychological detachment and emotion regulation skills. Work engagement, quality of life (physical health), absenteeism and presenteeism were not found to significantly differ between the iSMI and WLC groups. Changes in emotion regulation regarding general distress mediated changes in perceived stress. Conclusion The iSMI investigated in this study was found to be effective in reducing typical symptoms of stress. However, several important work-related health symptoms were not significantly affected by the intervention. Internet-based guided self-help interventions could be an acceptable, effective approach to reduce a range of negative consequences associated with work-related stress. Future studies should investigate the comparative (cost-) effectiveness of guided and unguided stress management interventions.