ABSTRACT Pancreatic stellate cells (PSCs) produce the stromal reaction of pancreatic cancer (PC) and their interaction with cancer cells facilitates cancer progression. This study investigated the role of human PSCs (hPSCs) in the metastatic process and tumor angiogenesis using an in vivo (orthotopic model) and in vitro (cultured PSC and PC cells) approach. A gender mismatch study [injection of male hPSCs + female PC cells into the pancreas of female mice] was conducted to determine whether hPSCs accompany cancer cells to metastatic sites. Metastatic nodules were examined by fluorescent in situ hybridization for the presence of the y chromosome. Angiogenesis was assessed by i) immunostaining tumors for CD31, an endothelial cell marker; and ii) in vitro quantifying human microvascular endothelial cell (HMEC-1) tube formation upon exposure to conditioned media from hPSCs. Transendothelial migration was assessed by examining the movement of fluorescently labeled hPSCs through an endothelial cell monolayer. Human PSCs i) were found in multiple metastatic sites in each mouse injected with male hPSCs + female PC cells; ii) increased CD31 expression in primary tumors from mice injected with MiaPaCa-2 and hPSCs and stimulated tube formation by HMEC-1 in vitro; iii) exhibited transendothelial migration which was stimulated by cancer cells. Human PSCs accompany cancer cells to metastatic sites, stimulate angiogenesis and have the capacity to intravasate/extravasate to and from blood vessels.