Dry eye syndrome (DES) is a common ocular disease that significantly affects the quality of life. Polyunsaturated fatty acids (PUFAs) have been used to treat DES; however, randomized controlled trials (RCTs) of PUFA therapy yield discordant results. The objective of this study was to clarify the effects of PUFAs on DES through meta-analysis of all relevant RCTs. To do so, a comprehensive search of PubMed, Embase, the Cochrane Library, ISI Web of Science, and unpublished data was conducted. The changes in clinical and laboratory examinations, symptomatic scores, and rates of relevant symptoms were analyzed. Nine RCTs were included in the current meta-analysis. Compared with placebo, PUFA supplementation was not related to changes in tear film break-up time (weighted mean difference [WMD], 0.33; 95% confidence interval [CI], -0.05 to 0.72), Schirmer's test score (WMD, 0.32; 95%CI, -0.23 to 0.86), or lissamine green staining score (WMD, -0.77; 95%CI, -1.66 to 0.12). However, significant reductions were detected in the symptom score on the ocular surface disease index (WMD, -2.26; 95%CI, -4.44 to -0.08) and in the rate of cells positive for human leukocyte antigen DR (WMD, -5.80; 95%CI, -8.62 to -2.97). This comprehensive meta-analysis supports the use of PUFA supplementation as a potential effective therapy for DES.