Objective: The pathogenesis of type 1 diabetes (T1D) is associated with genetic predisposition and immunological changes during presymptomatic disease. Differences in immune cell subset numbers and phenotypes between T1D patients and healthy controls have been described; however, the role and function of these changes in the pathogenesis is still unclear. Here we aimed to analyze the transcriptomic landscapes of peripheral blood mononuclear cells (PBMCs) during presymptomatic disease. Methods: Transcriptomic differences in PBMCs were compared between cases positive for islet autoantibodies and autoantibody negative controls (9 case–control pairs) and further in monocytes and lymphocytes separately in autoantibody positive subjects and control subjects (25 case–control pairs). Results: No significant differential expression was found in either data set. However, when gene set enrichment analysis was performed, the gene sets “defence response to virus” (FDR <0.001, ranking 2), “response to virus” (FDR <0.001, ranking 3) and “response to type I interferon” (FDR = 0.002, ranking 12) were enriched in the upregulated genes among PBMCs in cases. Upon further analysis, this was also seen in monocytes in cases (FDR = 0.01, ranking 2; FDR = 0.04, ranking 3 and FDR = 0.02, ranking 1, respectively) but not in lymphocytes. Conclusion: Gene set enrichment analysis of children with T1D-associated autoimmunity revealed changes in pathways relevant for virus infection in PBMCs, particularly in monocytes. Virus infections have been repeatedly implicated in the pathogenesis of T1D. These results support the viral hypothesis by suggesting altered immune activation of viral immune pathways in monocytes during diabetes.