To evaluate the effects of probiotic (VSL#3) and omega-3 fatty acid on insulin sensitivity, blood lipids, and inflammation, we conducted a clinical trial in 60 overweight (BMI > 25), healthy adults, aged 40-60 years. After initial screening the subjects were randomized into four groups with 15 per group. The four groups received, respectively, placebo, omega-3 fatty acid, probiotic VSL#3, or both omega-3 and probiotic, for 6 weeks. Blood and fecal samples were collected at baseline and after 6 weeks. The probiotic (VSL#3) supplemented group had significant reduction in total cholesterol, triglyceride, LDL, and VLDL and had increased HDL (P < 0.05) value. VSL#3 improved insulin sensitivity (P < 0.01), decreased hsCRP, and favorably affected the composition of gut microbiota. Omega-3 had significant effect on insulin sensitivity and hsCRP but had no effect on gut microbiota. Addition of omega-3 fatty acid with VSL#3 had more pronounced effect on HDL, insulin sensitivity and hsCRP. Subjects with low HDL, insulin resistance, and high hsCRP had significantly lower total lactobacilli and bifidobacteria count and higher E. coli and bacteroides count.