Background: Human parvovirus B19 (B19) infection can affect the hematopoietic arrest in fetus by hindering the differentiation and maturation of erythroid progenitor cells. B19 nonstructural protein 1 (NS1) has been shown to inhibit the differentiation of erythroid progenitor cells. The goal of this study is to explore the role of B19 NS1 in the regulation of GATA1 and Notch signaling pathway in hematopoietic cells. Methods: The B19 NS1 expression plasmid was reconstituted, and the possibility of NS1 regulating GATA1 and GATA2 expression modulated by Notch-Hes pathway was tested by qRT-PCR and western blot. Immunofluorescence assays were conducted to visualize pNS1 in K562 cells. Results: We demonstrate that B19 NS1 inhibited GATA1 and induced Hes1/Hes5, which is involved in the activation of Notch signaling pathway. Meanwhile, NS1 exhibited promoting effects on GATA2 expression. Activation of the Notch signaling pathway up-regulated its downstream transcriptional repressor family Hes, thereby inhibiting the expression of GATA gene in K562 cells. Conclusions: The results show that B19 NS1 protein negatively regulates GATA1 related nuclear transcription and may interfere with hematopoietic cell differentiation.