Pancreatic adenocarcinoma (PAAD) is a cancerous tumor with an extremely poor 5-year survival rate. The exploration of biomarkers for the diagnosis and treatment of PAAD is crucial in clinical practice. Krüppel-like factors (KLFs) are involved in a variety of biological functions in cells. According to multiple studies, KLF16 behave as an oncogene in prostate, breast and gastric cancers. However, no research has been done on the significance of KLF16 in PAAD.To explore the molecular mechanisms of KLF16 in PAAD.KLF16 was identified in the tumor specimens and normal tissues by GEPIA database and verified by quantitative real-time PCR (qRT-PCR). Knockdown or exogenous expression of KLF16, combined with in vitro and in vivo assays, was performed to show the functional significance of KLF16. The molecular mechanism of KLF16 was demonstrated by qRT-PCR, western blotting, immunoprecipitation assay and flow cytometry.We showed that KLF16 was highly expressed in PAAD patients based on the GEPIA database. KLF16 silencing suppressed while KLF16 overexpression promoted the malignant function of PAAD cells. Based on RNA sequencing, we discovered that KLF16 potentiated the expression of SMAD6 in PAAD cells. SMAD6 transcript abundance was increased and positively correlated with KLF16 expression in PAAD samples. In addition, inhibiting SMAD6 was able to mitigate the effects of KLF16 overexpression on PAAD cell processes, suggesting the importance of SMAD6 in the development of KLF16-triggered PAAD.KLF16/SMAD6 axis might be explored as a therapeutic target for PAAD therapy.©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.