Purpose:We sought to identify biomarkers that predict overall survival (OS) and response to immune checkpoint inhibitors (ICI) for patients with gastric cancer. Experimental design: This was a retrospective study of multiple independent cohorts of patients with gastric cancer. The association between tumor ACTA2 expression and OS and ICI response were determined in patients whose tumors were analyzed with bulk mRNA sequencing. Single-cell RNA sequencing (scRNA-seq) and digital spatial profiling data were used to compare tumors from patients with gastric cancer who did and did not respond to ICI. Results: Increasing tumor ACTA2 expression was independently associated with worse OS in a 567-patient discovery cohort [HR, 1.28 per unit increase; 95% confidence interval (CI), 1.02-1.62]. This finding was validated in three independent cohorts (n = 974; HR, 1.52 per unit increase; 95% CI, 1.34-1.73). Of the 108 patients treated with ICI, 56% of patients with low ACTA2 expression responded to ICI versus 25% of patients with high ACTA2 expression (P = 0.004). In an analysis of a publicly available scRNA-seq dataset of 5 microsatellite instability-high patients treated with ICI, the patient who responded to ICI had lower tumor stromal ACTA2 expression than the 4 nonresponders. Digital spatial profiling of tumor samples from 4 ICI responders and 5 ICI nonresponders revealed that responders may have lower ACTA2 expression in α-SMA-positive cancer-associated fibroblasts (CAF) than nonresponders (median: 5.00 vs. 5.50). Conclusions: ACTA2 expression is associated with survival and ICI response in patients with gastric cancer. ACTA2 expression in CAFs, but not in other cellular compartments, appears to be associated with ICI response.