The spontaneous immortalization of cells in vitro is a rare event requiring genomic instability, such as alterations in chromosomes and mutations in genes. In the present study, we report a spontaneously immortalized liver sinusoidal endothelial cell (LSEC) line generated from mouse liver. These immortalized LSECs showed typical LSEC characteristics with the structure of transcellular fenestrations, the expression of von Willebrand factor (VWF) and the ability to uptake DiI-acetylated-low density lipoprotein (DiI-Ac-LDL). However, these immortalized LSECs lost the ability to form capillary-like structures, and showed clonal and multilayer growth without contact inhibition. Moreover, their proliferation rate increased with the increase in the number of passages. In addition, these cells obained the expression of CD31 and desmin, and showed an upregulation of p53 protein expression; however, their karyotype was normal, and they could not form colonies in soft agar or tumors in SCID mice. In conclusion, in the present study, we successfully established a spontaneously immortalized LSEC line.