Lipedema is a chronic disorder affecting women with a 10% incidence worldwide. It is often confused with obesity. This study was undertaken to study microRNAs in lipedema tissue assessed by direct hybridization using the robust n-counter flex DX CE-IVD platform. The mean age of the subjects participating in the study was 40.29 (±12.17). The mean body weight and BMI were 67.37 (±10.02) and 25.75 (±4.10), respectively. The lipedema stages included were I and II. The differential expressed human (hsa)-miRNAs were determined according to a log2 fold-change (LFC) of 0.5 and p value < 0.05. To these, increased expression of hsa-let-7g-5p was evident, as well as reduced levels of hsa-miR-371a-5p, -4454+7975, -365a+b-3p, -205-5p, -196a-5p, -4488, -2116-5p, -141-3p, -208a-3p, -302b-3p, 374a-5p, and -1297. Then, several bioinformatics tools were used to analyze microarray data focusing on validated target genes in silico. KEGG and Gene Ontology (GO) pathway enrichment analysis was conducted. Furthermore, the protein–protein interaction and co-expression network were analyzed using STRING and Cytoscape, respectively. The most upregulated miRNA mainly affected genes related to cell cycle, oocyte meiosis, and inflammatory bowel disease. The downregulated microRNAs were related to endocrine resistance, insulin resistance, hypersensitivity to AGE-RAGEs, and focal adhesion. Finally, we validated by RT-PCR the upregulated hsa-let-7g-5p and two down-regulated ones, hsa-miR-205-5p and hsa-miR-302b-3p, confirming microarray results. In addition, three mRNA target miRNAs were monitored, SMAD2, the target of the hsa-let-7g-5p, and ESR1 and VEGFA, the target of hsa-miR-205-5p and hsa-miR-302b-3p, respectively. Our results open a new direction for comprehending biochemical mechanisms related with the pathogenesis of lipedema, shedding light on this intricate pathophysiological condition that could bring to light possible biomarkers in the future.