To investigate the speciation distribution characteristics of natural vitamin A in the liver of Epinephelus drummondhayi and its difference in improve effects on xerophthalmia compared with synthetic vitamin A, the saponification method combined with reverse-phase chromatography was used to determine the vitamin A components in the liver lipids of Epinephelus drummondhayi. F3, a fraction rich in natural vitamin A palmitate, was isolated from the neutral lipids. Using synthetic vitamin A palmitate as a control, the effects of natural vitamin A palmitate from Epinephelus drummondhayi liver on a benzalkonium chloride-induced xerophthalmia cell model and their differences were explored. The experiment included normal corneal epithelial cell group (NC), xerophthalmia cell model group (MC), F3 experimental group [low dose (LF3), medium dose (MF3), and high dose (HF3)], and vitamin A palmitate standard control group [low dose (LVAP), medium dose (MVAP), and high dose(HVAP)]. CCK-8 method was used to assess cell viability and toxicity, and real-time quantitative PCR was used to measure the gene expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in cells. The results showed that 99.57% of vitamin A in Epinephelus drummondhayi liver existed in a bound form, mainly as vitamin A palmitate, with the highest content in the liver neutral lipids. The F3 fraction, isolated and purified from the neutral lipids, contained natural vitamin A palmitate, and when the mass concentration of F3 exceeded 286.00pg/mL, the cell viability of HCE-T cells significantly decreased. Both F3 and synthetic vitamin A palmitate significantly improved the cell survival rate induced by benzalkonium chloride in the xerophthalmia cell model, reduced cell apoptosis, and the cell viability of the low and medium dose F3 group was higher than that of the high dose F3 group and all doses of synthetic vitamin A palmitate group. In terms of the relative expression level of interleukin (IL-6), the low and medium dose F3 group and the low dose synthetic vitamin A palmitate group were significantly higher than the medium and high dose synthetic vitamin A palmitate group(P<0.05). In the relative expression level of tumor necrosis factor-alpha (TNF-α), the low, medium, and high dose F3 groups were significantly lower than the low dose synthetic vitamin A palmitate group(P<0.05). Both F3 and synthetic vitamin A palmitate could downregulate the gene expression of IL-6 and TNF-α in HCE-T cells of the benzalkonium chloride-induced xerophthalmia cell model(P<0.05), alleviating the inflammation caused by xerophthalmia.