Abstract Context We previously demonstrated that insulin infusion altered metabolite concentrations in cerebral tissues assessed with proton magnetic resonance spectroscopy (1H-MRS) in young subjects with high insulin sensitivity, but not in those with low insulin sensitivity. Fat overload is an important factor leading to insulin resistance. Objective The purpose of the current study was to examine the effect of elevated circulating free fatty acid (FFA) levels on metabolites in cerebral tissues assessed with 1H-MRS. Design The study group comprised 10 young, healthy male subjects. 1H-MRS was performed at baseline and after 4-hour Intralipid (Fresenius Kabi)/heparin or saline infusions administered in random order. Voxels were positioned in the left frontal lobe, left temporal lobe, and hippocampus. The ratios of N-acetylaspartate (NAA), choline (Cho)-containing compounds, myo-inositol (mI), and glutamate/glutamine/γ-aminobutyric acid complex (Glx) to creatine (Cr) and nonsuppressed water signal were determined. Results Intralipid/heparin infusion resulted in a significant increase in circulating FFAs (P < 0.0001). Significant changes in brain neurometabolite concentrations in response to Intralipid/heparin infusion were increases in frontal mI/Cr (P = 0.041) and mI/H2O (P = 0.037), decreases in frontal and hippocampal Glx/Cr (P = 0.018 and P = 0.015, respectively) and Glx/H2O (P = 0.03 and P = 0.067, respectively), and a decrease in hippocampal NAA/Cr (P = 0.007) and NAA/H2O (P = 0.019). No changes in neurometabolites were observed during the saline infusion. Conclusions Acute circulating FFA elevation influenced cerebral metabolites in healthy humans and lipid-induced insulin resistance could be partly responsible for these effects.