Abstract: Objective To study the effect of gender and age on glycosylated hemoglobin level in healthy people in Chengdu. Methods A total of 41 795 healthy people were recruited in our study from 2009 to 2016. The average HbA1c levels of the healthy people were compared between different age groups and gender. Results The HbA1c levels increased with age in healthy people.The HbA1c levels in young, middle-aged and older group were 5.43%±0.58%, 5.75%±0.89%, 6.07%±0.98%, respectively in male, and 5.23%±0.36%, 5.56%±0.65%, 6.04%±0.93% respectively in female with statistically significant differences between age groups (All P< 0.01). The HbA1c levels were 5.32%±0.46%, 5.46%±0.61%, 5.64%±0.78%, 5.88%±0.97%, 6.04%±0.99%respectively in≤30 years, 31-40 years, 41-50 years, 51-65 years, > 65 years male groups, and 5.21%±0.36%, 5.23%±0.36%, 5.41%±0.54%, 5.71%±0.67%, 5.98%±0.92% respectively in≤ 30 years, 31-40 years, 41-50 years, 51-65 years, > 65 years female groups. The differences in HbA1c levels between male and female were statistically significant in all age groups (All P< 0.05). Conclusion The HbA1c level shows a gradual increasing trend with age, and it is higher in male compared with female in healthy people. We advise that the different reference value should be set by gender and age groups.