MicroRNAs (miRNAs) take part in tumorigenesis and show aberrant expression levels in cancerous tissues. We aimed to perform miRNA profiling of endometrioid endometrial cancer (EEC) metastatic loci derived from lymph nodes. Identification of aberrant miRNAs in positive lymph nodes could contribute to establishing new diagnostic markers and therapeutic targets. During the screening phase of the study, we performed profiling of 754 human miRNAs in endometrioid endometrial cancer tissues, microdissected metastatic loci from lymph nodes and healthy lymph nodes (Taqman Array). Selection of candidate miRNAs and subsequent validation using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 50 tissue samples were performed. After the screening phase of the study, five miRNAs were selected (hsa-miR-18b, hsa-miR-148a-5p, hsa-miR-204, hsa-miR-424, hsa-miR-129-1-3p). Validation revealed that miRNA-204 and miRNA-424 were highly downregulated in metastatic tissues compared with endometrial cancer samples (hsa-miR-204-P = .0008; hsa-miR-424-P = .0001). Receiver operating characteristic curves, which were constructed to compare endometrioid endometrial cancer and positive endometrioid endometrial cancer lymph nodes yielded the following area under the curves (AUCs): hsa-miR-204-.802 (96% confidence interval CI 0.676-0.927), hsa-miR-424-.84 (95% CI 0.711-0.969). Compared with primary endometrioid endometrial cancer tissue, metastatic loci derived from positive lymph nodes are characterized by profound downregulation of miRNA-204 and miRNA-424. © 2020 Nordic Federation of Societies of Obstetrics and Gynecology.