Oncolytic adenovirus could target and kill cancer stem cells and recognized as a promising anticancer agent. Our previous studies showed that oncolytic adenovirus ZD55 was developed to target liver cancer and exhibited obvious cytotoxicity effect. However, it still remained to be confirmed that whether ZD55 could also effectively eliminate liver cancer stem cells. We first utilized the suspension culture to enrich the liver cancer stem-like cells and validate the properties of acquired liver cancer stem cells. Further, we use MTT, Hoechst staining, Western blot, flow cytometry assay to detect cell viability, apoptotic effect and cytopathic effect in liver cancer stem-like cells after treatment with ZD55. The results indicated that liver cancer stem-like cells had the features of self-renewal, differentiation, high expression of cancer stem cell-related transcription factors (eg.NANOG and OCT4), quiescence, chemo-resistance. Oncolytic virus ZD55 resulted in obvious cytotoxicity and killing effect (the minimum cell viability for Huh7 sphere is 26.7%) on liver cancer stem-like cells, and induced significant cell apoptosis (the maximum apoptosis rate for Huh7 sphere is 60%). Thus, ZD55 might virtually represent an attractive therapeutic agent for targeting liver cancer stem cells to achieve better clinical outcome for HCC patients.