Objective To improve the production yield of N-glycosylated anti-VEGFR2 (vascular endothelial growth factor receptor 2) monobody (FN3(VEGFR2)-Gly) in lpp knockout Escherichia coli cells harboring Campylobacter jejuni N-glycosylation pathway. Results The leaky CLM37-Delta lpp strain efficiently secreted FN3(VEGFR2)-Gly into culture medium. The extracellular levels of glycosylated FN3(VEGFR2)-Gly in CLM37-Delta lpp culture medium were approximately 11 and 15 times higher compared to those in CLM37 cells via IPTG and auto-induction, respectively. In addition, the highest level of total glycosylated FN3(VEGFR2)-Gly (70 +/- 3.4 mg/L) was found in culture medium via auto-induction. Furthermore, glycosylated FN3(VEGFR2)-Gly was more stable than unglycosylated FN3(VEGFR2)-Gly in this expression system, but their bioactivities were relatively similar. Conclusions Lpp knockout leaky E. coli strain combined with auto-induction method can enhance the extracellular production of homogenous N-glycosylated FN3(VEGFR2)-Gly, and facilitate the downstream protein purification. The findings of this study may provide practical implications for the large-scale production and cost-effective harvesting of N-glycosylation proteins.