Abstract: Runx transcription factors are involved in hematopoiesis and differentiation, and play important roles in directed hematopoiesis in higher animals and insects, whereas the transcriptional regulation of Runx in the hematopoiesis in crustaceans is still unclear. To elucidate the regulatory role of Runx in hematopoiesis in crustaceans, a partial open reading frame (ORF) of the Runx transcription factor was cloned in this study, from Eriocheir sinensis and named as EsRunx. Semi-quantitative RT-PCR was employed to detect its tissue-specific expression. Hyalinocytes (HCs) and granulocytes (GCs) were isolated via Percoll gradient centrifugation, and real-time quantitative PCR (RT-qPCR) was used to analyze EsRunx expression differences between HCs and GCs. Dynamic expression changes of EsRunx in hematopoietic tissue (HPT) and hemocytes under exsanguination and lipopolysaccharide (LPS) stimulation were further examined. Additionally, dsRNA interference combined with flow cytometry was applied to investigate its impact on hemocyte differentiation. The partial ORF of EsRunx spans 650 bp, encoding 216 amino acids with a conserved Runt domain. Tissue expression analysis revealed specific high expression of EsRunx in hemocytes, with significantly higher levels in GCs than in HCs. Following exsanguination stimulation, EsRunx expression in HPT was significantly upregulated at 12 and 24 h, while in hemocytes, significant upregulation occurred at 2, 6, and 12 h. Under LPS stimulation, EsRunx expression in HPT increased markedly from 6 to 24 h, and in hemocytes, from 9 to 48 h. Silencing EsRunx gene led to a significant reduction in the percentage of medium and large granulocytes, while the percentage of HCs was notably increased. These findings demonstrate that EsRunx regulates hemocyte production in E. sinensis by promoting granulocyte differentiation. This study provides critical insights into the transcriptional regulatory mechanisms of hematopoiesis in crustaceans.