To stratify patients with mixed connective tissue disease (MCTD) based on their immunophenotype. We analyzed the immunophenotype and transcriptome of 24 immune cell subsets from patients with MCTD, systemic lupus erythematosus (SLE), idiopathic inflammatory myopathy (IIM), and systemic sclerosis (SSc) from our functional genome database, ImmuNexUT (https://www.immunexut.org/). MCTD patients were stratified by employing machine learning models including Random Forest, trained by immunophenotyping data from SLE, IIM, and SSc patients. Transcriptomes were analyzed with gene set variation analysis (GSVA) and clinical features of MCTD subgroups were compared. This study included 215 patients, including 22 patients with MCTD. Machine learning models, constructed to classify SLE, IIM, and SSc patients based on immunophenotyping, were applied to MCTD patients, resulting in 16 classified as SLE-immunophenotype and 6 as non-SLE-immunophenotype. Among MCTD patients, patients with the SLE-immunophenotype had higher proportions of Th1 cells 2.85% (interquartile range (IQR) 1.54-3.91) vs 1.33% (IQR 0.99-1.74) p= 0.027 and plasmablasts 6.35% (IQR 4.17-17.49) vs 2.00% (IQR 1.20-2.80) p= 0.010. Notably, the number of SLE-related symptoms was higher in patients with the SLE-immunophenotype 2.0 (IQR 1.0-2.0) vs 1.0 (IQR1.0-1.0) p= 0.038. Moreover, GSVA scores of interferon-α and -γ responses were significantly higher in patients with the SLE-immunophenotype in central memory CD8+ T cells, while hedgehog signalling was higher in non-SLE-immunophenotype patients in 5 cell subsets. This study describes the stratification of MCTD patients based on immunophenotyping, suggesting the presence of distinct immunological processes behind the clinical subtypes of MCTD.