MicroRNAs (miRs) are master regulators of post-transcriptional gene expression, and they are often dysregulated in individuals suffering from diabetes. We investigated the roles of miR-101-3p and miR-204-5p, both of which negatively regulate insulin secretion and cell survival and are highly expressed in pancreatic β cells, in the context of type 1 diabetes (T1D) pathogenesis. Using quantitative real time PCR, we evaluated serum levels of miR-101-3p and miR-204-5p in four groups, including recent-onset T1D patients (T1D group; = 50), individuals with normal glucose levels expressing one islet autoantibody (Ab) (single Ab group; = 26) or multiple autoantibodies (multiple Ab group; = 12), and healthy controls (control group; = 43). An analysis was performed to identify potential target genes of these miRNAs and to delineate enriched pathways. The relative expression of serum miR-101-3p was approximately three times higher in the multiple Ab and T1D groups than that in the single Ab and control groups ( < 0.0001). When considering all groups together, miR-101-3p expression was positively correlated with the level of islet autoantibodies GADA ( = 0.267; = 0.0027) and IA-2A ( = 0.291; = 0.001), and the expression of the miRNA was not correlated with levels of ZnT8A ( = 0.125; = 0.183). miR-101-3p expression did not correlate with HbA1c ( = 0.178; = 0.052) or glucose levels ( = 0.177; = 0.051). No significant differences were observed in miR-204-5p expression among the analyzed groups. Computational analysis of the miR-101-3p target gene pathways indicated a potential activation of the HGF/c-Met, Ephrin receptor, and STAT3 signaling pathways. Our study demonstrated that the circulating levels of miR-101-3p are higher in T1D patients and in individuals with normal glucose levels, testing positive for multiple autoantibodies, indicating that miR-101-3p precedes loss of glucose homeostasis. The pathogenic role of miR-101-3p in T1D may involve multiple molecular pathways.