BACKGROUND: Circulating microRNAs (c-miRs) were shown to be effective biomarkers for lung cancer early detection. However, the understanding of c-miRs origin and their biological functions still remains elusive. METHODS: We analysed miRNA expression in a large panel of lung cancer (LC) and hematopoietic cell lines (N = 252; CCLE database) coupled with c-miR profile of a large cohort of serum samples (N = 975), from high-risk subjects underwent annual LD-CT for 5 years. Furthermore, we examined intracellular and extracellular miR-29a-3p/223-3p expression profile in lung adenocarcinoma (LUAD) tissues, in matched serum samples and in LC and stromal cell lines. Lastly, through the modulation of expression of selected c-miRs by using mimic (OE) or antisense microRNA (KD), we explored their impact on lung cancer transcriptome and cancer and immune phenotypes. RESULTS: Here, we investigated the origin of an extensively validated 13 c-miRs signature diagnostics for asymptomatic lung cancer (LC) in high-risk subjects (smokers, >20 packs/y; >50 y old). Overall, we found a mixed origin of these c-miRs, originating both from tumour cells and the tumour microenvironment (TME). Intriguingly, we revealed that circulating miR-29a-3p and miR-223-3p are abundantly released from LC epithelial cells and immune cells, respectively. In particular, we found that miR-223-3p triggered several lung cancer related phenotypes such as invasion, migration and tumour-promoting inflammation. CONCLUSIONS: Our study highlights a mixed tumour epithelial and stroma-associated origin of LC c-miRs with new evidences on the multifaceted role of miR-223-3p in LC pathogenesis and immune modulation.