Membrane Protein Integrase is a glycolipid found in the inner membrane of Escherichia coli, playing an essential role in the integration of membrane proteins and the translocation of secretory proteins. Structurally, MPIase consists of 9–11 repeating trisaccharide units composed of N-acetyl-D-glucosamine, N-acetyl-D-mannosamineuronic acid, and 4-N-acetyl-D-fucosamine. The glycan chain is anchored to the membrane via a pyrophosphate-linked diacylglycerol moiety. Due to the limited availability and structural heterogeneity of natural MPIase, we undertook a systematic synthesis of MPIase analogs to elucidate its mechanism of action. Structure–activity relationship studies revealed that specific functional groups and glycan chain length critically influence membrane protein integration activity. Furthermore, we observed synergistic effects between MPIase analogs and the membrane chaperone YidC, along with chaperone-like activity inherent to the phosphorylated glycan portion. These findings provide insight into a translocon-independent pathway for membrane protein integration in E. coli, in which MPIase recognizes highly hydrophobic nascent proteins, prevents their aggregation, facilitates their recruitment to the membrane, and collaborates with YidC to promote successful integration, while concurrently regenerating its own activity.