Dendritic cells (DCs) as professional antigen presenting cells, are important in the initiation of the primary immune response. The present study compared the morphology, phenotypes and function between monocyte?derived human DCs produced from a conventional culturing system containing granulocyte?macrophage colony?stimulating factor (GM?CSF) and IL?4 (IL?4?DC) and DCs generated by the stimulation of GM?CSF and interferon (IFN)?α (IFN?DC). When compared with IL?4?DC in morphology, IFN?DC contained more organelles, including endoplasmic reticulum and myelin figures, whereas mature (m)IL?4?DC contained more vacuoles in the cells. The spikes of IFN?DC were shorter and thicker. The expression of phenotypes between immature IFN?DC and IL?4?DC were diverse. Following maturation with tumor necrosis factor?α, IFN?DC and IL?4?DC upregulated the expression of cluster of differentiation (CD)?11c and CD83. Conversely, immature IFN?DC and IL?4?DC secreted few inflammatory cytokines including interleukin (IL)?18, IL?23, IL?12p70, IL?1β and anti?inflammatory IL?10. Following maturation, large amounts of the cytokines were secreted by these two DCs and mIFN?DC secreted more cytokines compared with mIL?4?DC in general. Furthermore, immature IFN?DC and IL?4?DC loaded with cytomegalovirus (CMV)?pp65 protein were unable to induce the priming of T cells, as evaluated by the intracellular staining with IFN?γ. Notably, mature DCs exhibited the ability to present CMV?pp65 protein and activate T cells. The mIFN?DC activated a greater proportion of autologous CD4+ T cells (0.91 vs. 0.31%, P<0.001) and CD8+ T cells (0.90 vs. 0.48%, P<0.001) to secret IFN?γ compared with mIL?4?DC. The results suggested that the morphology, phenotypes and cytokine secretion of IFN?DC and IL?4?DC were diverse. The mIFN?DC were more effective in priming and cross?priming T cells when compared with IL?4?DC.