As a nutritional active protein in foods, multiple studies of the biological activities of lactoferrin had been proved, including antioxidant, antiviral, anti-inflammatory, antitumor, antibiosis and antiparasitic effects, while the mechanism related to its protection of cardiovascular system remained elusive. In the present work, the effect of lactoferrin on the viability of HUVECs (human umbilical vein endothelial cells) was detected to select the proper doses, the transcriptomics detection and data analysis were performed to screen out the special genes and the related pathway. Meanwhile, the regulation of lactoferrin in the functional factors thromboxane A₂ (TXA₂) and prostacyclin (PGI₂) was detected. Then, SiRNA fragment of the selected gene pyridoxal phosphatase (PDXP) was transfected into HUVECs to validate its role in protecting HUVECs function. Results showed that lactoferrin inhibited expression of TXA2 and activated expression of PGI2, as well as activated expression of PDXP, which significantly upregulated the synthesis of vitamin B₆ and the PI3K/AKT/ERK1/2 pathway. For the first time, we revealed that lactoferrin could induce the synthesis of vitamin B₆ and protect HUVECs function through activating PDXP gene and the related pathway.