Rationale Job stress can lead to job burnout, and BDNF polymorphism has been found to be involved in its psychopathological mechanism. Research needs a better understanding of the important role of gene x environment (i.e., BDNF polymorphism x job stress) interaction on job burnout. Objective This study aimed to explore how BDNF rs6265 polymorphism may moderate the relationship between job stress and job burnout. Methods Three hundred forty-one healthy participants (187 males and 154 females) from a Chinese university were included. The present study used a standardized questionnaire including demographic characteristics, job stress assessed by the House and Rizzo's Work Stress Scale, and job burnout assessed by the Maslach Burnout Inventory-General Survey. The BDNF rs6265 polymorphism was genotyped. Results Job stress showed a positive correlation with emotional exhaustion (p < 0.001), cynicism (p < 0.001), and reduced personal accomplishment (p < 0.01). The main effects of BDNF rs6265 polymorphism on emotional exhaustion and cynicism were significant [F-(1,F-333) = 5.136, p = 0.024; F-(1,F-333) = 4.175, p = 0.042, respectively]. The interaction between job stress and BDNF rs6265 on cynicism was significant (o R-2 = 0.013, p = 0.014) after controlling for age, sex, education, and position, indicating that individuals with BDNF rs6265 TT genotype showed higher level of cynicism when in high job stress. Conclusions The results provided evidence for the association of BDNF gene rs6265 polymorphism, job stress, and their interaction with job burnout. Individuals with TT genotype in BDNF rs6265 might be susceptible to stressful situations, which would lead to cynicism.