Gut microbiota have strong connections with health. Lactulose has been shown to regulate gut microbiota and benefit host health. In this study, the effect of short-term (3 week) intervention of lactulose on gut microbiota was investigated. Gut microbiota were detected from mouse feces by 16S rRNA high-throughput sequencing, and short chain fatty acids (SCFAs) were detected by gas chromatography-mass spectrometry (GC-MS). Lactulose intervention enhanced the alpha-diversity of the gut microbiota; increased the abundance of hydrogen-producing bacteria Prevotellaceae and Rikenellaceae, probiotics Bifidobacteriaceae and Lactobacillaceae, and mucin-degrading bacteria Akkermansia and Helicobacter; decreased the abundance of harmful bacteria Desulfovibrionaceae and branched-chain SCFAs (BCFAs). These results suggest that lactulose intervention effectively increased the diversity and improved the structure of the intestinal microbiota, which may be beneficial for host health.