Lymphatic metastasis of pancreatic cancer is a predictor of poor prognosis. However, the molecular mechanisms are largely unknown, thus, making the development of appropriate cell lines and experimental models critically important for future investigations. The purpose of the present study was to establish a 'pancreatic cancer cell and mouse model with high lymphatic metastasis potential' for in-depth study of the underlying mechanisms. The BxPC-3-LN subline, derived from the BxPC-3 human pancreatic cancer cell line, was established through serial passages in nude mice via footpad injections. The subline was able to develop notable lymphatic metastases in 100% of the recipient mice 8 weeks after tumor cell implantation. Compared with the parental BxPC-3 cells, BxPC-3-LN cells were more aggressive, displaying invasive ultrastructure, increased migration and invasion ability, and chemoresistance. Metastasis-related gene alteration including upregulation of MMP14, MMP24, MIF and ADRM1, and downregulation of TGFB2 and ROBO1 were also observed in BxPC-3-LN cells by cDNA microarrays. Thus, the newly selected BxPC-3-LN subline can serve as a unique model for further study of lymphatic metastasis of pancreatic cancer.