Objective To compare the phenotypes and biological characteristics between the rabbit hair follicle stem cells and urethral mucosa stem cells, and to explore the feasibility of using hair follicle stem cells as seed cells for tissue engineering urethra. Methods Rabbit hair follicle stem cells and urethral mucosa stem cells were isolated and cultured in vitro, and the cells with diameter <10 μm and integrin-α6+/CD71- were sorted using flow cytometry. The maximum amplification factor and clone forming ability of the two kinds of cells were calculated. The positive rates of K19, p63 and β1-integrin in the two kinds of cells were detected by flow cytometry. The enriched stem cells were implanted in the urethral stent to construct tissue engineering urethra. Histology and fluorescent staining were used to observe the tissue-engineered urethra. Results The maximum amplification multiples of rabbit hair follicle stem cells and urethral mucosa stem cells were (6.1 ±0.8)×10⁴ and (7.1±1.1)×10³, respectively; and the clone formation rates were (10.1±1.3)% and (4.3±1.1)%, respectively. The positive rates of K19, p63, and β1-integrin in hair follicle stem cells were (90.53±6.77)%, (93.31±5.57)%, and (91.17±6.98)%, respectively; and those in urethral mucosa stem cells were (88.50±4.95)%, (91.63±5.86)%, and (93.35±6.28)%, respectively. Both hair follicle stem cells and urethral mucosa stem cells formed complex layer epithelioid structure on the stents, with positive staining for AE1/AE3. Conclusion Rabbit hair follicle stem cells and urethral mucosa stem cells can both serve as seed cells for constructing tissue engineering urethra. As for the availability of seed cells, hair follicle stem cells are more superior to the urethral mucosa stem cells.