Objective To analyze the differences in cholesterol metabolism between male white hair and black eye (WHBE) rabbits and Japanese white rabbits by gut microbiota. Methods Six male WHBE rabbits and six male Japanese white (JW) rabbits aged 4 to 5 months were used and blood samples were collected for blood lipid detection. DNA was extracted from colon samples and 16S rRNA microbial sequencing was performed to observe changes in the structure and function of gut microbiota. Results Serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels of male WHBE rabbits were significantly lower than those of JW rabbits (P< 0. 05). Diversity analysis showed that the gut microbiota of these two strains of rabbits were significantly isolated, and the Chao1 index of WHBE rabbits was significantly lower than that of JW rabbits ( P< 0. 05). At the phylum level, the abundances of Fusobacteria, Tenericutes, and Cyanobacteria of WHBE rabbits were also lower than those of JW rabbits (P< 0. 05, P< 0. 01), and the Firmicutes/ Bacteroidetes ratio was lower than that of JW rabbits (P> 0. 05). At the genus level, the relative abundance of 24 genera in WHBE rabbits was significantly different from that of JW rabbits (P< 0. 05, P< 0. 01). Linear discriminant analysis effect size analysis showed that five key genera were found in WHBE rabbits, and Rikenelaceae_RC9_gut_group was the most abundant, whereas ten key genera were found in JW rabbits, and Ruminococcaceae_UCG_014 was the most abundant. PICRUSt functional prediction analysis showed that there were significant differences in arachidonic acid metabolism and ether lipid metabolism between gut microbiota in the two strains of rabbit (P< 0. 05, P< 0. 01). Correlation analysis showed that TC and LDL-C levels were positively correlated with Brevundimonas, Ileibacterium, Fusobacterium, Ralstonia and Mollicutes_RF39_unclassified, and negatively correlated with Coriobacteriaeae_unclassified, Anaerovorax,Clostridium,DTU014_unclassified, Barnesiellaceae_unclassified, Ruminococcaceae_UCG_009, and Clostridiales_vadinBB60_group_ unclassified. Conclusions The differences in cholesterol metabolism between male WHBE rabbits and JW rabbits may be related to differences in the structure and function of host gut microbiota, in which arachidonic acid metabolism and ether lipid metabolism pathways are particularly critical.