Background: Defensins are antimicrobial peptides that play important roles in innate immune response. Deregulation of beta-defensin-1 (DEFB1) gene expression has been implicated in several cancers and we previously showed that single nucleotide polymorphisms in DEFB1 are associated with clinical outcomes in patients with metastatic CRC. Hence, we aimed to further characterize the molecular features associated with DEFB1 gene expression in CRC. Methods: 14416 CRC tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or WES), RNA (WTS) and IHC were analyzed. Top quartile transcripts per million (TPM) for DEFB1 expression were considered high (Q4) while bottom quartile low (Q1). Consensus molecular subtypes (CMS) were assessed using RNAseq. Cell infiltration in the tumor microenvironment (TME) was estimated by QuantiSEQ. X²/Fisher-Exact were used and significance was determined as P-value adjusted for multiple comparison (Q<.05). Real-world overall survival information was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results:DEFB1 expression was highest in left-sided and rectal tumors (median TPM 2.27) and lowest in right-sided tumors (median TPM 1.62). Overall, when compared to low expression, high DEFB1 was negatively associated with high TMB (≥ 10 Mut/Mb) (4.8% vs 17%), dMMR/MSI-H (2.2% vs 13.1%), and PD-L1 expression (3.1% vs 5.2%) (all Q<.05). Additionally, DEFB1 high was associated with higher expression of immune checkpoint genes CD274, CD80, CD86, HAVCR2, LAG3, PDCD1 and PDCD1LG2 (Fold Change/FC: 1.27-1.56) but lower IDO1 (FC: 0.89) (all Q<.05). Similar results were confirmed in MSS tumors only, but IDO1 was now positively associated with DEFB1 high (FC: 1.23). In the MSS cohort, DEFB1 expression was highest in CMS2 and lowest in CMS3 (2.84 vs 1.67 median TPM, Q<.05). In the MSS cohort, APC mutations were more frequent in DEFB1 high tumors (79% vs 72%) while BRAF (5.8% vs 9.4%), GNAS (1.1% vs 4.4%), FBXW7 (7.8% vs 10.5%), SMAD4 (12.3% vs 17%), RNF43 (2.2% vs 3.5%) and POLE (0.2% vs 0.7%) mutations as well as MYC (1.2% vs 2.6%) and MYB amplifications (0.1% vs 0.9%) were less frequent in DEFB1 high (all Q<.05). Higher neutrophils, NK cells, M2 macrophages, CD4+ T cells and myeloid dendritic cells but lower M1 macrophages, Tregs and CD8+ T cells in the TME were significantly associated with high DEFB1; both in the overall and MSS cohorts (Q <.001). CRC patients with DEFB1 expression level above the median had worse OS compared to those below the median both in the overall cohort (HR: 1.18, 95% CI: 1.10-1.27) and in MSS tumors (HR: 1.18, 95% CI: 1.10-1.27). Conclusions: Our data show a distinct molecular landscape, including mutational profiles, CMS, immune biomarkers, and TME cell infiltration associated with DEF1B gene expression in CRC. These findings suggest a key role for DEF1B in modulating anti-tumor immunity and TME.