Abstract Over 80% of the patients with pancreatic ductal adenocarcinoma (PDAC) have cachexia, which is associated with reduced survival, decreased quality of life, and higher rates of metastatic disease. To date, the diagnostic and therapeutic options of PDAC-associated cachexia remain limited due to the lack of a complete understanding of the underlying pathological mechanism. Here, we demonstrate that fat loss is the earliest feature of cachexia induced by PDAC through its secreted exosomes. By analyzing exosomal components of normal and cancer-derived exosomes through small RNA-seq, we identify miR-16-5p, miR-21-5p, miR-29a-3p and miR-125b-5p enriched in pancreatic cancer-derived exosomes and in the exosomes from sera of mice or patients with pancreatic tumor. Among these, miR-16-5p and miR-29a-3p play a key role in fat loss by inhibiting adipogenesis through decreasing Erlin2 and CMPK1 expression leading to C/EBPβ and PPARγ downregulation. Synergistically, miR-29a-3p increases ATGL expression to facilitate lipolysis by suppressing MCT1 expression. Furthermore, PDAC-derived exosomes deprived of miR-16-5p and miR-29a-3p fail to induce fat loss, suggesting that these two exosomal miRs are required for PDAC-induced fat loss. These findings unravel a precise mechanism of how PDAC causes adipose atrophy via exosomal miRs and may provide new diagnostic and therapeutic strategies for PDAC-induced cachexia. Citation Format: Sui-Chih Tien, Chin-Chun Chang, I-Chih Chen, Ching-Hsuan Huang, Hsuan-Yu Peng, Yu-Ting Chang, Ming-Chu Chang, Chun-Mei Hu, Wen-Hwa Lee. Exosomal miRNA 16-5p/29a-3p secreted from pancreatic cancer induce adipose atrophy by inhibiting adipogenesis and promoting lipolysisExosomal miRNA 16-5p/29a-3p secreted from pancreatic cancer induce adipose atrophy by inhibiting adipogenesis and promoting lipolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 459.