Aim To investigate the effect of-94 ATTG insertion/deletion mutation at the promoter region of nuclear factor of kappa light polypeptide gene enhancer in B-cell 1 (NFKB1) gene on injury of human umbilical vein endothelial cell (HUVEC) induced by high glucose and high fat microenvironment and its potential mechanism. MethodsHigh glucose and high fat model of primary HUVEC cells with different genotypes of NFKB1 was established. Cellular apoptosis was detected by Annexin-V-FLUOS staining. Morphology of mitochondria was observed by laser confocal microscopy after Mito Tracker staining. Western blot was used to detect p50 and p65 proteins in nuclear factor-κB (NF-κB) signaling pathway, mitochondrial fission related proteins Drp1, Drp1-s637, Drp1-s616 and Fis1, mitochondrial fusion related proteins Mfn1, Mfn2 and Opa1, and apoptosis related protein cytochrome C (CytC). Results The apoptotic rate of primary HUVEC induced by high glucose and high fat was higher than control group, and the apoptotic rate of mutant type cells (DD genotype) was more than that of wild type cells (Ⅱ genotype). Mitochondria was over-fission into fragments.The expression of p50, a key protein in NF-κB pathway, was significantly decreased in DD genotype cells. Expression of mitochondrial fusion related protein Mfn2 was decreased. The phosphorylation of mitochondrial fission dynamic related protein Drp1-s616 was increased, and the expression of CytC was increased. Conclusion NFKB1 gene deletion mutation may be an important reason for the vulnerable damage of DD genotype HUVEC during high glucose and high fat microenvironment, and its potential mechanism may be related to mitochondrial over-fission.