Altered amino acid levels have been found in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). However, it is not clear whether this alteration is due to altered hepatic metabolism or insulin resistance. The aim of this study was to clarify the association among amino acid levels, fatty liver, and liver fibrosis while eliminating the influence of insulin resistance. NAFLD and liver fibrosis were diagnosed using transient elastography and subjects were divided in three groups: normal, NAFLD, and liver fibrosis. To exclude the influence of insulin resistance, the subjects were matched using the homeostasis model assessment of insulin resistance (HOMA-IR). The amino acid serum levels were compared among the groups. Of 731 enrolled subjects, 251 and 33 were diagnosed with NAFLD and liver fibrosis. Although significant differences were observed among the groups in the serum levels of most amino acids, all but those of glutamate and glycine disappeared after matching for HOMA-IR. The multivariate logistic regression revealed that glutamate, glycine, and HOMA-IR were independent risk factors for liver fibrosis. The altered serum levels of most amino acids were associated with insulin resistance, while the increase in glutamate and the decrease in glycine levels were strongly associated not only with insulin resistance, but also with altered liver metabolism in patients with liver fibrosis.