Objective: Currently, pharmaceutical treatment options for autism spectrum disorder are limited. Brain glutaminergic dysregulation is observed in autism spectrum disorder. N-acetylcysteine, which can be converted to glutathione and subsequently release glutamate into the extracellular space, and thus reduce glutamatergic neurotransmission at synapses, is considered a potential drug for autism spectrum disorder treatment. Here, we analyzed the treatment effects of N-acetylcysteine on autism spectrum disorder in randomized controlled trials. Study design: Updated systematic review and meta-analysis. Data sources: By systematically searching the PubMed, Embase and Cochrane Library, we obtained five randomized controlled trials. Study selection: Meta-analyses were performed to examine the improvement in autistic behaviors as measured by the Aberrant Behavior Checklist, Social Responsiveness Scale and Repetitive Behavior Scale–Revised, using mean difference with a 95% confidence interval and a random-effects model. Data Synthesis: After 8–12 weeks of N-acetylcysteine supplementation, the pooled result of four trials revealed an improvement in Aberrant Behavior Checklist total score (mean difference = 1.31, 95% confidence interval = [0.42, 2.20]). When one trial was excluded, the sensitivity test result was stronger (mean difference = 1.88, 95% confidence interval = [0.92, 2.83]). The pooled results of three trials revealed significant improvements in hyperactivity (mean difference = 4.80, 95% confidence interval = [1.20, 8.40]) and irritability (mean difference = 4.07, 95% confidence interval = [1.13, 7.04]). Regarding Social Responsiveness Scale, the pooled result of two trials showed significant improvement in social awareness after 8–12 weeks of N-acetylcysteine supplementation (mean difference = 1.34, 95% confidence interval = [0.09, 2.59]). No differences were observed in the pooled results of two trials using Repetitive Behavior Scale, either in the total or the subscales. Conclusion: We concluded that N-acetylcysteine is safe and tolerable, reduces hyperactivity and irritability and enhances social awareness in children with autism spectrum disorder. However, further evidence should be sought before a general recommendation.