The phytochrome-interacting factors (PIFs) function crucially in multiple physiological processes, but the biological functions of some PIFs remain elusive in some species. Here, a tobacco (Nicotiana tabacum L.) PIF transcription factor NtPIF1 was cloned and characterized. The transcript of NtPIF1 was significantly induced by drought stress treatments, and it localized in the nuclear. Knockout of NtPIF1 by CRISPR/Cas9 system led to the improved drought tolerance of tobacco with increased osmotic adjustment, antioxidant activity, photosynthetic efficiency and decreased water loss rate. On the contrary, NtPIF1-overexpression plants display drought-sensitive phenotypes. Moreover, NtPIF1 reduced carotenoids biosynthesis and regulated the expression of carotenogenic genes upon drought stress. Electrophoretic mobility shift and dual-luciferase assays illustrated that NtPIF1 directly binds to the promoter of the carotenogenic gene Ntß-LCY to repress its transcription. Overall, these data suggested that NtPIF1 negatively regulates the tobacco adaptive response to drought stress and carotenoids biosynthesis; moreover, NtPIF1 has the potential to develop drought-tolerant tobacco plants using CRISPR/Cas9 system.