Background and Aims: Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of the metabolic syndrome and is strongly linked with obesity and type 2 diabetes. The role of gut-liver interaction is increasingly recognised in the development of NAFLD. Modification of gut microbiota may lower cardiovascular risk and reduce liver injury beyond existing treatment in those with NAFLD. This study tests the hypothesis that probiotic supplementation may improve endothelial function and insulin sensitivity; and reduce oxidative stress, inflammation and liver injury in subjects with NAFLD. Methods: This is a randomised, double-blinded, placebo-controlled, proof-of-concept trial in which subjects with NAFLD are allocated to take either two sachets VSL#3 probiotic twice daily or the placebo equivalent for 10 weeks. Biophysical markers for endothelial function, oxidative stress, vascular inflammation, insulin resistance and liver injury were undertaken before and after the intervention period. Results Forty-two patients participated and 35 of them completed the study. There were 28 males and 7 females; and 74% had type 2 diabetes or impaired fasting glycaemia. Mean age was 57 ± 8 years, body mass index 32.6 ± 5.0 kg/m2, blood pressure 134/82 ± 13/7 mmHg, HbA1c 53 ± 14 mmol/mol (7.0 ± 3.4%), total cholesterol 4.42 ± 1.15mmol/l, HDL 1.06 ± 0.29mmol/l, LDL 2.43 ± 1.06 mmol/l, triglycerides 2.00 ± 0.88 mmol/l, ALT 53 ± 26 iu/l and AST 40 ± 15 iu/l. Median duration of NAFLD was 0.3 ± IQR 2.0 years. No significant difference was seen in markers of cardiovascular risk and liver injury following VSL#3 probiotic supplementation. Conclusion: There was no significant improvement in the markers of endothelial function, oxidative stress, inflammation, insulin resistance, liver fibrosis scores, liver transaminases or liver imaging in this group of patients with NAFLD treated with 10 weeks of VSL#3 probiotic supplementation. The results may be due to a number of factors such as a small sample size, subjects with relatively good metabolic control and possibly less severe liver disease, and the lack of consensus on an effective dose and duration of probiotic supplementation.