Objective：To construct and identify the recombinant adenovirus AdNICD1 carrying Notch1 intracellular domain（NICD1）and observe the influence of the overexpression of AdNICD1 on the activation of Notch signaling pathway. Methods：NICD1 coding area was amplified by high fidelity PCR（Hi-Fi PCR）and then subcloned to recombinant adenovirus vector by Gibson assembly. Bacteria screening and gene sequencing were used to make sure NICD1 was subcloned to recombinant adenovirus vector accurately. And then the plasmid was transfected into BJ5183 to complete homogenous recombination and generate recombinant adenovirus plasmid pAdNICD1. pAdNICD1 was identified by plasmid screening and linearized by PacⅠ enzyme，and then transfected to human embryo kidney 293 cell line（HEK293）for packaging. The packaged AdNICD1 were collected and amplified stepwisely. Immortalized multipotent adipose-derived mesenchymal stem cells（iMAD）were infected by AdNICD1，and the infection efficiency was tested. QPCR and Western blot were used to detect the expression of NICD1，and the downstream gene expression of Notch signaling pathway was determinated by QPCR. Results：NICD1 was successfully subcloned to the recombinant adenovirus vector，and AdNICD1 was successfully packaged and amplified in HEK293 cell line. The overexpression of NICD1 was confirmed by QPCR and Western blot in MSCs. Notch signaling pathway downstream gene expressions were also upregulated by AdNICD1. Conclusion：The recombinant adenovirus AdNICD1 was successfully constructed，and overexpression of NICD1 can exogenously activate Notch signaling pathway.