(1) Background: Wharton’s Jelly stem cells (WJ-MSCs) are multipotent mesenchymal stem cells that can proliferate rapidly and have low immunogenicity. Therefore, WJ-MSCs have gained considerable attention in the fields of immunomodulation and disease treatment and have entered clinical trials for the treatment of various diseases. Therefore, it is crucial to study the underlying mechanisms of WJ-MSCs proliferation, immune regulation, and disease treatment. Nuclear Receptor Subfamily 2 Group F Member 2 (NR2F2) is a transcription factor that is involved in the regulation of many different genes. However, it remains unknown how NR2F2 regulates stem cell identity in WJ-MSCs. (2) Methods: We used RNAi technology to knock down NR2F2 in WJ-MSCs, and studied the regulatory role of NR2F2 in WJ-MSCs by MTT, flow cytometry, RNA-seq, and other methods. We also utilized a co-culture system in which NR2F2-depleted WJ-MSCs with MH7A and HCT116/HepG2 were used to investigate the role of NR2F2 in immunomodulation and the inhibition of cancer cell growth. (3) Results: NR2F2 knockdown resulted in decreased expressions of Cyclin D1 and CDK4, slower cell proliferation, and increased expressions of IL6 and IL8. Furthermore, Cyclin D1, CDK4, and inflammatory factors were increased in human rheumatoid fibroblast-like synoviocyte line MH7A if co-cultured with NR2F2 depleted WJ-MSCs. In addition, we observed increased p53, decreased BCL-2, and increased cell apoptosis in liver cancer cell line HepG2 if co-cultured with NR2F2-depleted WJ-MSCs. (4) Conclusions: NR2F2 not only plays an important role in the cell cycle and immune regulation of WJ-MSCs but also has potential effects on the WJ-MSCs treatment of related diseases.