This study aimed to assess the impact of yeast single-cell protein (YP) supplementation in diets from late gestation through lactation on sow reproductive performance and the associated gut microbiome and metabolomic changes in milk. A total of 172 sows, at 103 days of gestation, were randomly assigned to four treatment groups: a control group receiving 2% fishmeal and three groups receiving 0.5%, 1.0%, and 2.0% YP, replacing 0.3%, 0.6%, and 1.5% fishmeal, respectively. No significant effects were observed on litter performance in sows. The inclusion of 2% YP displayed an augmented litter weight gain and piglet weight gain during lactation. Microbial sequencing revealed a marked decrease in Enterobacteriaceae abundance in sow feces at day 113 of gestation following dietary YP supplementation. Moreover, it led to a notable reduction of microbial-associated lipids, such as endotoxin, in serum and milk. In summary, YP supplementation in sow diets reduced gut pathogenic microbiota and their components, contributing to enhanced growth performance in suckling piglets.