Long-term administration of methotrexate (MTX) for management of chronic inflammatory diseases is associated with risk of liver damage. In this study, we examined the transcriptional profiles of livers from patients treated with MTX. The possibility that expression signatures correlate with grade of fibrosis or underlying rheumatic disease was evaluated. Twenty-seven patients taking MTX were accrued for this study. Ten non-MTX-exposed normal liver specimens were used as controls. Global mRNA expression was assayed using oligonucleotide arrays. A total of 205 genes were significantly altered in MTX-exposed livers. Six of these genes were validated by qPCR. Two genes, CLN8 and ANKH that map to chromosomal locations previously associated with rheumatoid arthritis, were found to be elevated in MTX-exposed samples. Subsequent pathway analysis indicates that MTX exposure is associated with the following key alterations: (1) upregulation of lipid biosynthetic genes, consistent with MTX-induced steatosis, (2) downregulation of proinflammatory chemokines, consistent with the anti-inflammatory effects of MTX, and (3) elevation of complement pathway gene expression. Complement 5, shown earlier to be correlated with liver fibrosis in mice, was found to be elevated (twofold) in MTX-exposed livers. In conclusion, we have found the expression of a number of genes associated with rheumatic disease and/or MTX exposure to be significantly different. Differences in complement expression provide the rationale for future correlative studies between MTX-induced liver fibrosis and C5 alleles in order to identify patients with increased risk for fibrosis.