Objective: There have been multiple reports of the success of using 5% imiquimod to treat hemangioma of infancy (HOI) lesions. There have been no published studies looking at the effect of imiquimod on these lesions on a molecular level. Therefore, we investigated the effect of imiquimod in a human endothelial cell line (HUVEC). Study Design: Basic science laboratory experiments Methods: Human umbilical vein endothelial cells (HUVEC) were plated and treated with different concentrations of imiquimod. After treatment was complete, media was collected and the cells were harvested for RNA and protein isolation. ELISA, reverse transcriptase PCR, and western blots were carried out using the harvested media, RNA, and protein respectively, and comparisons between treatment groups were made. Furthermore, cell proliferation in HUVEC cells treated with imiquimod was also evaluated using MTT assays. Results: In the HUVEC cells, there was a dose dependent decrease in cell proliferation with imiquimod treatment based on MTT assays. However, there were no significant differences in IL-8 or VEGF secretion by ELISA, reverse transcriptase PCR revealed no difference in expression in IL-8, VEGF, Ki-67, MMP-9, Cyclin D1, Interferon-alpha, or Caspase-3, and western blot also showed no difference in amounts of Cyclin D1, Caspase-3, PARP, and PCNA between the treatment groups. Conclusions: Treatment of a HUVEC cell line with imiquimod resulted in decreased cell proliferation in a dose dependent response based on MTT analysis, however we were unable to identify a potential pathway for this as there was no difference in a number of molecular studies between the treatment groups.