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Background Formula feeding is a well-established risk factor for necrotizing enterocolitis (NEC) in preterm infants. Although prior studies have implicated intestinal dysbiosis as a key contributor to NEC pathogenesis, the precise mechanisms underlying formula-induced NEC remain incompletely understood. This study aims to identify probiotic strains suitable for incorporation into infant formula to prevent NEC and to elucidate their protective mechanisms of action. Methods NEC rats and HT29 cells were used to screen for probiotics with anti-inflammatory effects. Hematoxylin-eosin staining and ELISA were used to assess the inhibitory effect of LRX01 on intestinal pyroptosis in a rat model of NEC. Western Blot and qPCR were employed to analyze the levels of inflammatory responses and the expression of related proteins, and 16S rRNA sequencing was utilized to assess the changes in the intestinal microbiota.Results We found that formula milk feeding significantly increased the proportion of Gram-negative bacteria and activated the TLR4/GSDMD/IL-1βpathway of the colon, resulting in an increase in pyroptosis in NEC rats. In vitro and in vivo experiments, Lactobacillus rhamnosus LRX01 was screened from the feces of healthy infants, for the good anti - inflammatory effects and probiotic properties. The results showed Lactobacillus rhamnosus LRX01 significantly inhibited the TLR4/GSDMD/IL-1βpathway, lowered the intestinal inflammation and pyroptosis in NEC rats, and its effect was superior to Lactobacillus rhamnosus GG. Even if probiotics were only supplemented before model establishment, they still exerted this protective effect. Furthermore, 16S rRNA sequencing revealed that LRX01 could modulate the intestinal microbiota, increase the abundance of Lactobacillus and Gram-positive bacteria in the intestine, and restore the intestinal microbiota imbalance in NEC.Conclusions Preventive supplementation with Lactobacillus rhamnosus LRX01 significantly increased intestinal microbiota abundance and alleviated intestinal pyroptosis induced by formula feeding in NEC rats.