Increasing evidence supports that cancer stem cells (CSCs) are responsible for driving tumor initiation and maintenance. Zinc-finger E-box binding homeobox 1 (ZEB1) is a transcription factor for regulating tumor progression, and contributes to maintenance of CSC-like properties. The goal of the present study is to investigate the effect of decreasing ZEB1 expression on the B16F10 CSC-like properties. The recombinant shRNA targeting ZEB1 were transfected into melanoma B16F10 cells, and shZEB1-CD133(+)CD44(+) CSCs were isolated from the stable transfected cells using the magnetic-associated cell sorting method. The shZEB1-CD133(+)CD44(+) CSC-like properties were systematically analyzed. The results show the B16F10 shZEB1-CD133(+)CD44(+) CSCs significantly decreased the ability of clonogenicity, cellular proliferation, migration, and invasion. Importantly, tumorigenicity and tumor lung metastasis was significantly inhibited in B16F10 shZEB1-CD133(+)CD44(+) CSCs compared with B16F10 scramble-CD133(+)CD44(+) CSCs. The decrease of ZEB1 expression markedly resulted in down-regulation of vimentin and N-cadherin expression as well as up-regulation of E-cadherin expression in tumor tissues from the mice injected with B16F10 shZEB1-CD44(+)CD133(+) CSCs. These findings contribute to understanding the maintenance of B16F10 CD133(+)CD44(+) CSC-like properties that was closely associated with ZEB1 expression. ZEB1 may serve as a new therapeutic target for treatment of malignant melanoma.