To compare the efficacy, prognosis, and toxicity of S-1-based with fluorouracil (5-FU)-based chemotherapy in patients with advanced gastric cancer (AGC) as first-line treatment, we performed this meta-analysis of all eligible randomized controlled trials (RCTs). A comprehensive literature search of electronic databases (up to February 20, 2014) was performed. Additionally, abstracts presented at the American Society of Clinical Oncology (ASCO) conferences held between January 2000 and February 2014 were searched to identify relevant trials. Overall response rate (ORR), time to treatment failure (TTF), overall survival (OS), and grade 3 or 4 toxicities were analyzed. Six RCTs with 2,264 patients of AGC were included. Meta-analysis demonstrated that S-1-based therapy was associated with better OS compared with 5-FU-based therapy (hazard ratio (HR) = 0.80, 95 % confidence interval (CI) 0.80-0.99, P = 0.03). Pooled estimate has showed the trend of superiority of S-1-based therapy in the aspect of ORR (odds ratio (OR) = 1.55, 95 % CI 0.87-2.77, P = 0.14) and TTF (HR = 0.73, 95 % CI 0.53-1.00, P = 0.05), but the difference was not significant. The incidence of toxicities of 5-FU-based regimens was significantly higher for thrombocytopenia (OR = 0.60, 95 % CI 0.42-0.88, P = 0.008) and stomatitis (OR = 0.22, 0, 95 % CI 0.05-0.9, P = 0.03). Based on the published studies, S-1-based therapy was superior to 5-FU-based therapy in OS and safety profile as first-line treatment in AGC. It was prone to improving ORR and TTF, though the difference was not significant. More high-quality randomized controlled trials should be performed to provide more information in comparing these two regimens.