Objective Many studies have reported that long-term exposure to job-related stress can lead to burnout, which may be influenced by genetic and environmental factors. Burnout correlates with depression. This study investigated whether one tag polymorphism rs6354 in 5-HTT gene modulated the influence of job-related stress on burnout in the medical professionals in a Chinese Han population, which to our best knowledge has not been explored. Methods Seven hundred twelve subjects were recruited from a general hospital and measured for burnout symptoms using the Maslach Burnout Inventory (MBI), the stress using the House and Rizzo's Work Stress Scale, and the stressors using the Evers, Frese, and Cooper's Sources of Pressure Scale. The 5-HTT rs6354 polymorphism was genotyped in 376 subjects. Results The majority of correlations between the work stress score or the six stressor scores and three burnout subscores were significant (all p < 0.05). There was no significant main effect of the 5-HTT rs6354 genotype on burnout symptoms; however, there was a statistically significant interaction between 5-HTT rs6354 and work stress on burnout (F = 5.08, df = 2, 369, p = 0.007). In the low stress group, G allele carriers had significantly higher burnout level than TT homozygote (F = 11.60, df = 1, 48, p < 0.001). On the contrary, in the high stress group, G allele carriers exhibited significantly lower burnout level compared to TT homozygote (F = 3.86, df = 1, 103, p = 0.025). Conclusions Our findings suggest that the 5-HTT rs6354 polymorphism may modulate the influence of job-related stress on burnout by adjusting serotonin transporter function and neurotransmission, showing that individuals with TT genotype displayed a greater susceptibility to both the detrimental effects of higher stress and the beneficial effects of lower stress compared to those with G allele, which supports the differential-susceptibility hypothesis.