Purpose: The piggyBac transposon system is known to non-viral integrate exogenous genes in the chromosomes of mammalian cells. This study exploited the reporter gene imaging, mediated by the piggyBac transposon system, to track the growth and dissemination of 4T1 triple-negative murine breast cancer cells in vivo. Subsequently, the study conducted an ex vivo analysis of the metastatic cells expressing reporter genes. Methods: A PB-3R-puro construct harboring multiple cistrons was used for piggyBac-mediated reporter genes imaging. These cistrons include monomeric red fluorescence protein (mRFP), luciferase 2 (luc2), and herpes simplex virus type I—thymidine kinase (HSV1-tk). In addition, 4T1 cells stably transfected with PB-3R-puro construct were implanted to Balb/C mice to form synergistic tumors. Further, liver metastatic 4T1 tumor was visualized by bioluminescent imaging. It was also excised to analyze cancer stem cell-related characteristics, including sphere formation, drug resistance, CD44 expression, and tumorigenesis. Results: The piggyBac transposon system did not influence the proliferation rate, invasion and migration rate, and mammosphere formation ability of 4T1 cells. The liver metastatic cells, named 4T1-3R_L cells, exhibited cancer stem cells (CSC)-related characteristics compared to parental 4T1 cells. We also found that 4T1-3R_L cells exhibited stronger migration and invasive abilities by wound healing assay and in vitro invasion assay, respectively. The microarray assay showed that epithelial–mesenchymal transition (EMT)-promoting markers, including vimentin, N-cadherin, Twist1, and Snail, were up-regulated, and the anti-EMT marker E-cadherin was down-regulated in 4T1-3R_L cells. Conclusion: Current data suggest that the piggyBac transposon system can engineer cancer cells for tacking and characterizing tumor development in vivo and ex vivo.