Cardiovascular disease (CVD) is the leading cause of death in the United States. Recently, the gut microbiota has been implicated in the pathophysiology and progression of CVD. Experimental evidence suggests that high fat feeding alters the functional composition of the gut microbiota (dysbiosis); leading to increased translocation of the pro-inflammatory, endotoxin, and increased production of the pro-atherogenic, trimethylamine-N-oxide (TMAO). Together, these changes are hypothesized to accelerate CVD progression. Conversely, administration of gut microbiota modulating agents, such as antibiotics and probiotics, attenuate high fat feeding induced CVD in rodent models. In humans, the capacity to produce TMAO following L-carnitine or phosphatidylcholine challenges is abolished after receiving broad spectrum antibiotics for a period of one week. However, whether gut modulation over a longer period of time decreases fasting serum endotoxin, fasting plasma TMAO, and CVD risk in response to high fat feeding has been unexplored in humans. To address these issues we conducted a randomized, placebo controlled, parallel group designed, controlled feeding study in healthy, non-obese males receiving the multi-strain probiotic, VSL #3 (or placebo), while a consuming a high fat diet for 4-weeks. First, we tested the hypothesis that VSL #3 would attenuate the rise in serum endotoxin and consequent arterial stiffening following high fat feeding in healthy, non-obese males. Second, we tested the hypothesis that VSL #3 would attenuate the rise in plasma TMAO concentrations following high fat feeding in healthy, non-obese males. In contrast to our first hypotheses, serum endotoxin concentrations and arterial stiffness did not change in response to high fat feeding or with VSL#3 treatment. Interestingly, VSL #3 significantly attenuated the increase in body mass (+ 1.4±0.4 vs. +2.3±0.3 kg; P < 0.05) and fat mass (+0.7±0.1 vs. + 1.4±0.3 kg; P < 0.05) following high fat feeding compared to the placebo. In contrast to our second hypothesis, probiotic supplementation did not attenuate the rise in plasma TMAO following high fat feeding. Future studies are necessary to elucidate the mechanisms responsible for the prevention of body mass and fat mass gain with VSL#3 supplementation following high fat feeding. In addition, studies are needed to determine whether higher doses of VSL #3, other single or multispecies probiotics, prebiotics, or synbiotics attenuate the production of the proatherogenic, TMAO.