Abstract Purpose: This study performed a comprehensive molecular characterization of microsatellite instability–high (MSI-H)/mismatch repair–deficient (dMMR) gastrointestinal (GI) tumors to elucidate predictors of response to PD-1 blockade. Experimental Design: Forty-five patients with MSI-H/dMMR GI tumors, including gastric cancer, colorectal cancer, cholangiocarcinoma, small intestine cancer, pancreatic cancer, and duodenal cancer, receiving PD-1 blockade were analyzed. We conducted the genomic profiling of GI tumors by whole-exome sequencing or targeted next-generation sequencing. The tumor microenvironment was evaluated by transcriptomic analysis and multiplex fluorescence IHC. Results: Patients with low tumor mutational burdens (TMBs) had lower objective response rates (ORRs; 0% vs. 48.8%) and a significantly shorter progression-free survival (PFS; 2.3 vs. 15.6 months; HR, 6.20; P = 0.002) than those with high TMBs. Among common gene alterations in GI tumors, only PTEN mutations, which were mutually exclusive with a low TMB, were significantly associated with a lower ORRs than wild-type PTEN (21.4 vs. 54.8%; odds, 4.45; P = 0.045). Compared with wild-type PTEN, PTEN mutations in the phosphatase domain were associated with significantly lower ORRs (12.5 vs. 54.8%; P = 0.049), shorter PFS (2.6 vs. 15.6 months; HR, 5.04; P < 0.001), lower intratumoral CD8+ T-cell levels, higher intratumoral CD204+ macrophage levels, and PI3K/AKT/mTOR pathway enrichment, whereas PTEN mutations in the C2 domain were not. Conclusions: Low TMBs and PTEN mutations, especially mutations in the phosphatase domain associated with an immunosuppressive environment, were mutually exclusive and might be negative predictors of PD-1 blockade responses in patients with MSI-H/dMMR GI tumors.