Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most aggressive solid tumours in humans. Despite its high mortality rate, effective targeted therapeutic strategies remain limited due to incomplete understanding of the underlying biological mechanisms. The NAP1L gene family has been implicated in the development and progression of various human tumours. However, the specific function and role of NAP1L5 (nucleosome assembly protein-like 5) in PDAC have not been fully elucidated. Therefore, in this study, we aimed to investigate the role of NAP1L5 in PDAC and explore the regulatory relationship between NAP1L5 and its potential downstream molecule PHLPP1 (PH domain Leucine-rich repeat Protein Phosphatase 1) in PDAC. Our study revealed that NAP1L5 is notably upregulated in PDAC. Moreover, both in vivo and in vitro experiments demonstrated that knockdown of NAP1L5 suppressed the proliferation of PDAC cells. Mechanistically, NAP1L5 was found to promote PDAC progression by activating the AKT/mTOR signalling pathway in a PHLPP1-dependent manner. Specifically, NAP1L5 binds to PHLPP1 and facilitates the ubiquitination-mediated degradation of PHLPP1, ultimately resulting in reduced PHLPP1 expression. Notably, TRIM29, recruited by NAP1L5, was found to be involved in facilitating K48-linked ubiquitination of PHLPP1. Our findings indicate that NAP1L5 overexpression promotes the proliferation of PDAC cells by inhibiting PHLPP1 expression. These novel insights suggest that NAP1L5 may serve as a potential therapeutic target for PDAC.Copyright © 2023. Published by Elsevier Inc.