BACKGROUND: CD73 and CD39, key components of the adenosine axis, are expressed in multiple malignancies; the impact of standard-of-care treatment on their expression and antitumor immunity in esophageal squamous cell carcinoma (ESCC) remains unclear. We evaluated the adenosine axis in the context of neoadjuvant therapy received and its relationship to immune markers in ESCC tumor samples. METHODS: Samples from patients who underwent surgical resection at the National Cancer Center Hospital, Tokyo, Japan, between January 2002 and July 2019 following no neoadjuvant therapy (n = 55; treatment-naïve), chemotherapy (n = 200), or chemoradiotherapy (CRT; n = 20) were immunohistochemically stained for CD73, CD39, PD-L1, FoxP3, and CD8; markers were quantified across tumor microenvironment (TME) compartments. RESULTS: Median CD73 TME expression was lower in the treatment-naïve (2.8%) versus chemotherapy (7.2%; p < 0.0001) and CRT (6.4%; p < 0.01) cohorts, most profoundly in the stroma (median 4.1% vs 9.4% [p < 0.0001] and 8.1% [p < 0.01]). Median intraepithelial CD8-positive cell density was higher in the treatment-naïve (200.7 cells/mm(2)) versus chemotherapy (93.9 cells/mm(2); p < 0.0001) and CRT (30.5 cells/mm(2); p < 0.001) cohorts. Three-year recurrence-free survival (RFS) was 73.0%, 58.0%, and 30.0%, and 3-year overall survival (OS) was 78.2%, 71.4%, and 33.5%, in the treatment-naïve, chemotherapy, and CRT cohorts, respectively. High versus low CD73 TME expression was prognostic for longer RFS (treatment-naïve cohort: hazard ratio [HR] 0.16, 95% confidence interval [CI] 0.05-0.58, p = 0.0014; chemotherapy cohort: HR 0.52, 95% CI 0.34-0.78, p = 0.0012) and OS. CONCLUSIONS: These translational data demonstrating higher CD73 expression in tumors after neoadjuvant chemotherapy or CRT support potential combination strategies with CD73-targeted treatment in ESCC.