Background: Acceptance and commitment therapy (ACT) is an empirically supported transdiagnostic approach that involves mindfulness processes and behavior change processes for valued living. Objective: This systematic review and meta-analysis of randomized controlled trials (RCTs) aimed to assess the efficacy of internet-based ACT (iACT) for depressive symptoms, anxiety, stress, psychological distress, and quality of life (QoL). Methods: PubMed, CINAHL, PsycINFO, and SCOPUS databases were searched to identify relevant RCTs published up to June 5, 2021. The included RCTs were assessed using the Cochrane Collaboration risk-of-bias tool. The use of either a random effects model or fixed effects model was determined using I2 statistic values for heterogeneity. Subgroup analyses were conducted according to the type of control group, the use of therapist guidance, delivery modes, and the use of targeted participants, when applicable. Results: A total of 39 RCTs met the inclusion criteria. Meta-analyses found small effects of iACT on depressive symptoms, anxiety, stress, psychological distress, and QoL at the immediate posttest and follow-up. There was no significant effect of iACT on stress at follow-up. Subgroup analyses showed small to medium effects of iACT on all the outcomes at the immediate posttest and follow-up compared with the passive control groups. In contrast, subgroup analyses that compared iACT with active control groups found no differences between groups on stress, psychological distress, and QoL at the immediate posttest or on depressive symptoms, anxiety, and stress at follow-up. In addition, subgroup analyses conducted according to the use of therapist guidance, delivery modes, and the use of targeted participants found no statistically significant subgroup differences among studies in all the outcomes, except for the subgroup difference among studies according to the use of targeted participants for depressive symptoms at the immediate posttest (ie, a statistically significant, larger effect of iACT when studies targeted people with depressive symptoms). The overall risk of bias across the studies was unclear. Conclusions: The findings of this study contribute to the body of evidence regarding the effects of iACT on depressive symptoms, anxiety, stress, psychological distress, and QoL and may be applicable in any population, as ACT is a transdiagnostic approach. Few studies have compared iACT with active control conditions, especially for stress and psychological distress at the immediate posttest and follow-up. In addition, the active control conditions varied among the included studies. Further high-quality studies are needed to better understand whether iACT is comparable or superior to other evidence-based interventions, such as cognitive behavioral therapy, in decreasing depressive symptoms, anxiety, stress, and psychological distress and improving QoL.