Abstract: Polar growth in higher plants is very common, but the relationship between vesicle trafficking and polarity growth is not completely clear. Root hairs and pollen tubes are two typical models of polar cell growth in plants. Previous studies have shown that NtGNL1 (Nicotiana tabacum GNOM-LIKE 1) plays an important role in tobacco pollen tube elongation by regulating post-Golgi trafficking. Accordingly, we conducted further detailed investigation on NtGNL1 function in root hair tip growth based on NtGNL1 RNAi transgenic lines. Results demonstrated that root hair growth was obviously obstructed in NtGNL1 RNAi transgenic lines in contrast with the control. The NtGNL1 RNAi root hair phenotypes, such as tip swelling, bending, and curving, were similar to those in pollen tubes of NtGNL1 RNAi transgenic plants. Using qRT-PCR, the mRNA expressions of the root hair growth polarity genes in NtGNL1 RNAi transgenic lines were detected, including PIN1, PIN2, GL2, ROP6, and RHD6. Results showed the expressions of PIN2 and GL2 were downregulated significantly, while the expressions of PIN1, RHD6, and ROP6 did not change obviously. Observation of vesicle distribution in the root epidermis cells and root hairs by FM4-64 revealed that the vesicles accumulated inside the cytosol, suggesting that NtGNL1 affected vesicle trafficking in root hairs. Furthermore, BFA treatment intensified the accumulation, implying that other BFA-sensitive genes were involved in root hair growth. In summary, NtGNL1 influenced tip growth of root hair via the vesicular trafficking pathway. Also, downregulation of NtGNL1 reduced the expressions of PIN2 and GL2, thus indirectly affecting root hair polarity.