Abstract: In order to explore the function of KNOX family genes in tobacco leaf development, a KNOX family gene NtabKNOX8 was cloned from Nicotiana tobacum cv. Honghuadajinyuan and bioinformatics analysis was performed. Transgenic technique was performed to obtain NtabKNOX8 over-expression (OE) lines. And then GUS staining experiments were performed on the leaves of the hybrids (F1) of materials transferred into the auxin reporter DR5::GUS and materials overexpressing NtabKNOX8. The results showed that NtabKNOX8 belongs to the KNOX family subfamily Ⅰ. Compared to the wild type tobacco plants, the NtabKNOX8 OE lines showed round and wrinkled leaves which curved toward the back and lost leaf symmetry. Meanwhile, the midrib of the two OE lines was greatly shortened and the venation changed significantly to palmately-netted venation-like type, with the leaf shape index and the leaf area decreased, leaf specific fresh weight increased. In addition, the auxin content was significantly increased while its distribution was more widely and extensive in the tobacco leaves overexpressing NtabKNOX8. It is suggested that NtabKNOX8 may regulate the growth and development of tobacco leaves by affecting the content and distribution of auxin, which finally leads to multiple changes in tobacco leaves.