This study aims to explore how a high-fat diet and glutaredoxin1 (Glrx1) deficiency affect the development of obesity in male and female mice. A high-fat diet induced great differences in calorie intake and body weight gain between male and female mice; furthermore, the Glrx1 deficiency made male mice more sensitive to a high-fat diet than females. Male mice had higher glucose intolerance, and Glrx1 deficiency aggravated gender differences in glucose intolerance. Glrx1 deficiency aggravated high-fat diet-induced hyperlipidemia. The mRNA levels of HMGCR, Srebf-1c, Srebf-2, CD36, FASN and SCD1 were consistently lower in females than in males. Glrx1 deficiency exacerbated high-fat diet induced liver injury and oxidative stress. Diet but not gender or genotype altered the composition of gut microbiota. These findings provide a new insight into the different susceptibilities to obesity caused by a high-fat diet between males and females.