Epidermal growth factor (EGF) ameliorates stress and prevents incomplete gastrointestinal development in early-weaned piglets in commercial swine farming. This study aimed to further analyze the biological activities of intracellularly expressed EGF (IE-EGF), extracellularly expressed EGF (EE-EGF), and tagged EGF (T-EGF) from Saccharomyces cerevisiae in early-weaned pigs. In this study, we assigned 24 pigs to each of 5 groups that were provided a basic diet (the control group) or a diet supplemented with empty vector-expressing S. cerevisiae [the INVSc1(EV) group], T-EGF-expressing S. cerevisiae [the INVSc1-TE(-) group], EE-EGF-expressing S. cerevisiae [the INVSc1-EE(+) group], or IE-EGF-expressing S. cerevisiae [the INVSc1-IE(+) group]. All treatments were delivered at a dose of 60 μg EGF/kg body weight (BW) everyday. All the piglets were sacrificed after 21 day to determine their physio-biochemical indexes, immune functions, and intestinal development. In the piglet experiments, recombinant S. cerevisiae survived throughout the intestinal tract. The BW and intestinal development (e.g., mean villous height, crypt depth, villous height:crypt depth ratio (IVR), and total protein, DNA, and RNA contents) of the piglets were significantly enhanced in the INVSc1-IE(+) group compared with the animals in the INVSc1-EE(+) and INVSc1-TE(-) groups (P < 0.05). In addition, increased proliferating cell nuclear antigen (PCNA) staining was observed in the piglets that received the INVSc1-IE(+) treatment (approximately 80 %) compared with those that received the INVSc1-TE(-) (approximately 70 %) and INVSc1-EE(+) treatments (approximately 70 %). The levels of lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALP), immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) were also significantly increased in the INVSc1-IE(+) group compared with the INVSc1-EE(+) and INVSc1-TE(-) groups (P < 0.05). Furthermore, the proliferation of piglet enterocytes was also significantly stimulated by both IE-EGF and EE-EGF compared with T-EGF in vitro (P < 0.05). Our data further demonstrate the previously reported hypothesis that IE-EGF is more suitable than EE-EGF or T-EGF for applications in early-weaned pigs.