Background: Chronic exposure to job-related stress can lead to depression and BDNF polymorphism may play an important role in this process. The role of the stress x BDNF Val66Met interaction in depression has been studied widely using childhood stress, but few studies have utilized chronic stress in adulthood as a moderator. This study was to examine the chronic stress x BDNF Val66Met interaction in job-related depression in the healthcare workers in a Chinese Han population, which has not been reported yet. Methods: Using a cross-sectional design, 243 doctors and nurses were recruited from a general hospital in Beijing, and were assessed for depression with Self-rating Depression Scale (SDS), and the stress using the House and Rizzo's Work Stress Scale. The BDNF Val66Met polymorphism was genotyped. Results: There was a significant positive association between job stress and depressive scores (p < 0.001). No significant main effect of the BDNF Val66Met genotype on depressive symptoms was observed (p > 0.05). A statistically significant interaction between BDNF Val66Met and job stress on depressive symptoms was found (p < 0.05); individuals with Val/Val genotype showed a higher SDS score than Met allele carriers only in the low-stress group, without significant differences in SDS score between the BDNF Val66Met subgroups in medium-or high-stress group. Limitations: Limitations include cross-sectional study design, the small sample size only in healthcare workers and only one polymorphism in BDNF gene was analyzed. Conclusions: Our results suggest a close relationship between job-related stress and depression, and the interaction of the BDNF Val66Met polymorphism and chronic stress in adulthood may impact the depressive symptoms.