The acquisition of neuroendocrine (NE) characteristics by prostate cancer (PCa) cells is closely related to tumour progression and hormone-resistance. The mechanisms by which NE cells influence PCa growth and progression are not fully understood. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in oncogenic processes, and MIF serum levels correlate with aggressiveness of PCa. Here, we investigated the regulation and the functional consequences of MIF expression during NE transdifferentiation of PCa cells. NE differentiation of LNCaP cells, initiated either by increasing intracellular levels of cAMP or by culturing cells in an androgen depleted media, was associated with markedly increased MIF release. Yet, intracellular MIF protein and mRNA levels and MIF gene promoter activity decreased during NE differentiation of LNCaP cells, suggesting that NE differentiation favours MIF release despite decreasing MIF synthesis. Adenoviral-mediated forced MIF expression in NE differentiated LNCaP cells increased cell proliferation without affecting the expression of NE markers. Addition of exogenous recombinant MIF to LNCaP and PC-3 cells stimulated the AKT and ERK1/2 signalling pathways, the expression of genes involved in PCa, as well as proliferation and resistance to paclitaxel and thapsigargin-induced apoptosis. Altogether, these data provide evidences that increased MIF release during NE differentiation in PCa may facilitate cancer progression or recurrence, especially following androgen deprivation. Thus, MIF could represent an attractive target for PCa therapy.