This experiment was conducted to explore the effects of gut microbiota on neonatal diarrhea in a germ-free (GF) pig model. Twelve hysterectomy-derived GF piglets were housed in six sterile isolators. Among them, six piglets were treated as the GF group, and the other six piglets were orally introduced with healthy sow fecal suspension and regarded as the fecal microbiota transplantation (FMT) group. Another six piglets from natural birth were considered as the conventional (CV) group. The GF and FMT piglets were hand-fed with sterile milk powder for 21 days, and the CV piglets were suckled for the same days. Then, all piglets were fed with sterile feed for another 21 days. Results exhibited that the GF group's fecal score and moisture level were higher than those in the CV and FMT groups ( < 0.05). Meanwhile, the abundances of colonic AQP1 and AQP8 in the GF group were the greatest among these treatments ( < 0.05). However, FMT piglets had a lower fecal score in d 22-28 and d 29-35 than that in the CV piglets ( < 0.05). Collectively, the absence of gut microbiota may cause diarrhea in the piglet model, and transplantation of maternal fecal microbiota may reverse it.