Rapamycin (RAPA) as a unique tolerance-promoting therapeutic drug is crucial to successful clinical organ transplantation. DC (Dendritic cells) play a critical role in antigen presentation to T cells to initiate immune responses involved in tissue rejection. Although the influence of RAPA on DC differentiation and maturation had been reported by some research groups, it is still controversial and unclear right now. In addition, it is also lack of study on investigating the role of DC in DTH reaction via intravital optical imaging. Herein, we investigated the effect of rapamycin on phenotype and function of bone marrow monocyte-derived DC both in vitro and in vivo. In vitro experiments by flow cytometry (FACS) showed that DC displayed decreased cell size and lower expression levels of surface molecule CD80 induced by RAPA; Furthermore, the phagocytic ability to OVA of DC was inhibited by RAPA started from 1 h to 2 h post co-incubation, but recovered after 4 h; In addition, the capacity of DC to activate naïve OT-II T cell proliferation was also inhibited at 3 day post co-incubation, but had no effect at 5 day, the data indicated this effect was reversible when removing the drug. More importantly, the DC-T interaction was monitored both in vitro and in intravital lymph node explant, and showed that RAPA-DC had a significant lower proportion of long-lived (>15min) contacts. Thus, RAPA displayed immunosuppressive to phenotypic and functional maturation of DC, and this phenomenon induced by RAPA may favorable in the clinical organ transplantation in future.