Context: Most epigenetic studies in diabetes compare normal cells in "high glucose" (HG) to cells in "normal glucose" (NG) and cells returned from HG to NG. Here we challenge this approach. Objective: The objective was to determine whether there were differences in gene expression in skin fibroblasts of monozygotic twins (MZT) discordant for type 1 diabetes (T1D). Design: Skin fibroblasts were grown in NG (5.5 mmol/L) and HG (25 mmol/L) for multiple passages. Setting: This study was conducted at the University of Minnesota. Patients: Patients were nine MZT pairs discordant for T1D. Main Outcome Measure(s): Gene expression was assessed by mRNA-Seq, using the Illumina HiSeq 2000 instrument. Pathway analysis tested directionally consistent group differences within the Kyoto Encyclopedia of Genes and Genomes pathways. Results: A total of 3308 genes were differentially expressed between NG and HG in T1D MZT vs 889 in non-T1D twins. DNA replication, proteasome, cell cycle, base excision repair, homologous recombination, pyrimidine metabolism, and spliceosome pathways had overrepresented genes with increased expression in T1D twins with P values ranging from 7.21 × 10-10 to 1.39 × 10 × -4. In a companion article, we demonstrate that these pathway changes are related to diabetic nephropathy risk. There were no pathways statistically significant differently expressed in nondiabetic twins in HG vs NG. Conclusions: In vivo exposure to diabetes alters cells in a manner that markedly changes their in vitro responsestoHG. These results highlight the importance of using cells directly derived from diabetic patients for studies examining the effects of HG in diabetes.