Objetive To construct shRNA-hnRNPU lentiviral expression vector and stable hnRNPU-knockdown （KD） murine preB A70 cell line. Methods shRNA against mouse hnRNPU gene was encapsulated in pLKO.1-EBFP-PURO vector. Identified by sequencing，pLKO.1-hnRNPU shRNA-EBFP-PURO vector was co-transfected with lentiviral packaging plasmids into HEK293T cells to form lentiviruses，and the lentiviral supernatant was collected. The packaged lentiviruses transduced A70 cells which were observed by an inverted fluorescence microscope. Western blot was used to test hnRNPU expression. Single cell clones were constructed by limiting dilution，stable hnRNPU-KD A70 cell line was formed and identified by Western blot. Results The shRNA-hnRNPU lentiviral expression vector was successfully constructed，and the lentiviruses expressing shRNA-hnRNPU were packaged in HEK293T cells. 72 h after transduction，blue fluorescence was observed in the virus-infected A70 cells. HnRNPU expression in puromycin-treated cells decreased about a half. By limiting dilution，hnRNPU-KD cell lines were formed. Conclusion In this study，a stable hnRNPU knockdown A70 cell line was successfully constructed，which laid the experimental foundation for further study of the role of hnRNPU in early B cell development.