Pancreatic stellate cells (PSCs) are the precursors of cancer-associated fibroblasts (CAFs), which potentiate pancreatic tumor growth and progression. In this study, we investigated whether Lipoxin A4 (LXA4), an endogenous bioactive lipid, can inhibit the differentiation of human PSCs (hPSCs) into CAF-like myofibroblasts and thereby hPSC-induced pro-tumorigenic effects. LXA4 significantly inhibited TGF-β-mediated differentiation of hPSCs by inhibiting pSmad2/3 signalling. Furthermore, treatment with LXA4 abolished the paracrine effects (proliferation and migration of Panc-1 tumor cells) of hPSCs in vitro. These data demonstrated that LXA4 can interrupt pro-tumoral paracrine signalling of hPSCs. Furthermore, LXA4 treatment significant decreased the size and growth rate of 3D-heterospheroids comprised of hPSC and Panc-1 and these effects were exhibited due to inhibition of hPSC-induced collagen1 expression. In vivo, we examined the therapeutic efficacy of LXA4 in a co-injection (Panc-1 and hPSCs) subcutaneous tumor model. Intriguingly, LXA4 significantly abolished the tumor growth (either injected intratumor or intraperitoneally), attributed to a significant reduction in fibrosis, shown with collagen1 expression. Altogether, this study proposes LXA4 as a potent inhibitor for hPSCs which can be applied to reprogram tumor stroma in order to treat pancreatic cancer.