To investigate the effects of 1,3-dioleoyl-2-palmitoyl-glycerol (OPO) on the gut health of weanling mice, we fed weanling mice for four weeks, classified them into three groups: the control group (CON group), the low-dose OPO (LOPO) group, and the high-dose OPO (HOPO) group, and compared the groups. Various indicators were analyzed, such as serum inflammation levels, intestinal tissue morphology, gene expression of tight junction proteins, intestinal permeability markers, gut microbiota diversity, and changes in short-chain fatty acids (SCFAs). The results showed that, compared with the CON group, serum levels of TNF-α and IL-1β decreased by 9.05% (P<0.01) and 9.71% (P<0.05), respectively, in the LOPO group and 10.71% (P<0.001) and 7.84%, respectively, in the HOPO group. The relative mRNA expression of claudin-1 increased by 31.37% (P<0.05) in the LOPO group when compared with the CON group, whereas the value increased by 38.24% (P<0.001) in the HOPO group. Additionally, serum LPS levels in the LOPO and HOPO groups decreased by 8.43% and 8.66% (P<0.05), respectively, compared with the CON group. Furthermore, dietary OPO regulated the gut microbiota composition, increasing the relative abundance of Akkermansia, Dubosiella, and Bifidobacterium and decreasing the relative abundance of Desulfovibrio. SCFA results indicated that, compared with the CON group, the contents of acetic acid, propionic acid, butyric acid, and valeric acid increased by 162.32%, 72.7%, 216.24%, and 25.2%, respectively, in the LOPO group and by 160.68%, 74.58%, 163.85%, and 23.03%, respectively, in the HOPO group (P<0.001). In conclusion, dietary OPO may enhance intestinal barrier function and inhibit systemic inflammatory responses in weanling mice by modulating gut microbiota and related SCFA metabolism. These findings provide a theoretical basis for targeted regulation of gut microbiota by OPO.