Liposomes (LPs) as promising drug delivery systems are widely applied in cancer therapy. This study aimed to investigate the effect of glypican-1 (GPC1)-targeted and gemcitabine (GEM)-loaded LP [GPC1-LP (GEM)] on cell proliferation and apoptosis in PANC-1s, as well as on orthotopic pancreatic cancer (PDAC) mice. The GPC1-LP (GEM) and LP (GEM) was prepared, and then the size distribution of GPC1-LP (GEM) was analyzed by dynamic light scattering (DLS). In vitro drug release assay of GPC1-LP (GEM) and LP (GEM) was performed, and the expression of GPC1 in PANC1 cells was detected as well. Next, the effects of free GEM, LP (GEM) and GPC1-LP (GEM) on cell viability, clone number, and apoptosis, as well as the expression of proteins associated with apoptosis were measured in 239T and PANC-1 cells. Furthermore, the body weight and tumor size of orthotopic PDAC mice were evaluated following the treatment of free GEM, LP (GEM) or GPC1-LP (GEM). LP (GEM) and GPC1-LP (GEM) were successfully prepared with a successful GEM release within 24 h. In addition, GPC1 was positively expressed in PANC-1 cells but not 293T cells. These findings provided more insights into the anti-tumor potential for the biomedical application of GPC1-LP (GEM) in PDAC.