Although preclinical studies clearly indicate an effect of 5-HTTLPR genotype on 5-HT transporter (5-HTT) expression, studies in humans provided inconclusive results, hypothetically due to environmental factors and differences in individual behavior. For example, nicotine and other constituents of tobacco smoke elevate serotonin (5-HT) levels in the brain and may thereby cause homeostatic adaptations in 5-HTT availability that moderate effects of 5-HTTLPR genotype. To test whether 5-HTT availability in the midbrain is affected by smoking status and 5-HTTLPR genotype, we pooled data from prior studies on in vivo 5-HTT availability (BP) measured with positron emission tomography (PET) and [C]DASB. In total, we reanalyzed 5-HTT availability in 116 subjects using ANCOVA statistics. ROI analysis revealed that current smokers and non-smokers do not differ in midbrain BP. Interestingly, smoking status significantly interacted with 5-HTTLPR genotype: active smoking was associated with reduced 5-HTT availability only in LL subjects but not in carriers of the S-allele. From the perspective of genotype effects, non-smokers showed the expected association with 5-HTTLPR, i.e. higher 5-HTT availability in LL subjects compared to carriers of the S-allele, whereas this pattern was actually reversed for active smokers. Our study indicates that smoking status moderates the association of 5-HTTLPR genotype and 5-HTT expression, which may help to explain inconsistent findings in previous studies. Regarding the mechanism, we suggest that smoking may induce epigenetic processes such as methylation of SLC6A4, which can differ depending on its genetic constitution.