Purpose <p>Chemotherapy-induced neutropenia is a serious adverse event. Combination chemotherapy with docetaxel, cisplatin, and 5-fluorouracil (DCF) is an effective neoadjuvant therapy for esophageal cancer, but it is associated with a high incidence of Grade 4 neutropenia. In previous study, we identified associations between genetic variants and severe neutropenia. The aim of this study was to explore genetic factors related to pharmacokinetics and endogenous antioxidant defense mechanisms that may be associated with the development of Grade 4 neutropenia in esophageal cancer patients treated with DCF chemotherapy using two independent cohorts.</p> Methods <p>We conducted a retrospective pharmacogenetic analysis using DNA samples from the National Cancer Center Biobank in Japan. Two independent cohorts of esophageal cancer patients treated with the DCF chemotherapy were analyzed: an exploratory cohort (n = 157) and a validation cohort (n = 121). Single nucleotide polymorphisms (SNPs) in genes related to docetaxel pharmacokinetics and the Keich-like ECH-associated protein 1-Nuclear factor erythroid2-related factor 2 (Keap1-Nrf2) antioxidant system were examined.</p> Results <p>A total 53 (33.8%) and 62 (51.2%) patients developed Grade 4 neutropenia in exploratory and validation cohort, respectively. Multivariate analysis revealed that age, <i>Adenosine triphosphate-binding cassette (ABC) G2</i> rs2231137G &gt; A and Nuclear factor, erythroid 2 like 2 (<i>NFE2L2</i>) rs35652124C &gt; T were significant in exploratory cohort, while baseline absolute neutrophil count and <i>NFE2L2</i> rs35652124C &gt; T were significant in the validation cohort.</p> Conclusions <p><i>NFE2L2</i> rs35652124C &gt; T was identified as an independent predictive genetic factor for Grade 4 neutropenia in esophageal cancer patients treated with DCF chemotherapy.</p>