Low positive cell screening efficiency severely hinders the development of transgenic animals. The major rate-limiting step of positive cell screening is DNA entering the nucleus, particularly for large DNA molecules. To enhance the transport of large DNA molecules into the nucleus, particularly for the production of transgenic animals, nuclear localization sequence (NLS) peptides and the peptide derivative succinimidyl-[4-(psoralen-8-yloxy)]-butyrate (SPB)-NLS were synthesized to mediate transfection in vitro. To investigate the function of NLS and SPB-NLS in vitro, the expression levels of growth hormone (GH) mRNA and green fluorescent protein (GFP) protein were analyzed following transfection mediated by NLS and SPB-NLS. The results demonstrated that the expression of GH mRNA was significantly higher in the NLS (increased by 69%) and SPB-NLS (330%) groups than that in the liposome/pGN group. Similarly, GFP expression was found to be higher in the SPB-NLS group than that in the liposome group, while the expression in the NLS group was lower than that in the liposome group. Further analysis demonstrated that SPB-NLS enhanced the expression of insulin-like growth factor 1 in hard-to-transfect goat mammary epithelia cells. The results of the microscopy analysis revealed that transfected DNA entered the nucleus via the nuclear pores, facilitated by NLS. Analysis of the cell cycle demonstrated that the cytotoxic effects of NLS and SPB-NLS were low. In conclusion, the results of the present study demonstrate that SPB-NLS acts as a transfection-enhancing agent and may be used both to enhance nuclear delivery and for the development of genetically modified animals.