Background Lack of knowledge of systematic reviews (SRs) could prevent individual health care professionals from using SRs as a source of information in their clinical practice or discourage them from participating in such research. Objective In this randomized controlled trial, we evaluated the effect of a short web-based educational intervention on short-term knowledge of SRs. Methods Eligible participants were 871 Master’s students of university health sciences studies in Croatia; 589 (67.6%) students who agreed to participate in the trial were randomized using a computer program into 2 groups. Intervention group A (294/589, 49.9%) received a short web-based educational intervention about SR methodology, and intervention group B (295/589, 50.1%) was presented with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) checklist. The participants’ knowledge of SRs was assessed before and after the intervention. The participants could not be blinded because of the nature of the intervention. The primary outcome was the difference in the percentage of correct answers about SR methodology per participant between the groups after the intervention, expressed as relative risk and 95% CI. Results Results from 162 and 165 participants in the educational intervention and PRISMA checklist groups, respectively, were available for analysis. Most of them (educational intervention group: 130/162, 80.2%; PRISMA checklist group: 131/165, 79.4%) were employed as health care professionals in addition to being health sciences students. After the intervention, the educational intervention group had 23% (relative risk percentage) more correct answers in the postintervention questionnaire than the PRISMA checklist group (relative risk=1.23, 95% CI 1.17-1.29). Conclusions A short web-based educational intervention about SRs is an effective tool for short-term improvement of knowledge of SRs among health care studies students, most of whom were also employed as health care professionals. Further studies are needed to explore the long-term effects of the tested education. Trial Registration OSF Registries 10.17605/OSF.IO/RYMVC; https://osf.io/rymvc