Background: Macrophages play a crucial role in the interaction between tumor and immune system, and iNOS is known as a surrogate marker of M1 macrophages activation. The goal of the study was to investigate the role of iNOS polymorphisms as prognostic marker in mCRC patients. Materials and methods: Functional significant polymorphisms in the promoter of INOS gene were analyzed by PCR-based and direct DNA sequencing in 4 cohorts of patients receiving bevacizumab based first-line chemotherapy: two evaluation cohorts (TRIBE ARM A and ARM B) and two validation cohorts (FIRE 3 arm A and MOMA). The relation of the SNPs with PFS and OS was evaluated through Kaplan-Meier method and log-rank test. Subgroup analyses according to RAS status were preplanned. Results: In the exploratory cohort 1 (TRIBE A), patients with CCTTT any> 13repeats (N = 57) showed improved median PFS compared with patients carrying the <= 13/<= 13 repeats variant (N = 152) (HR, 0.64;95% CI 0.44-0.92, p = 0.010). Similar results were shown adopting the >26repeats/<= 26 repeats (HR, 0.56;95% CI 0.36-0.87, p = 0.005). In RAS mutant, patient with any> 13 repeats (N = 24) had improved PFS results compared with those carrying the <= 13/<= 13 repeats variant (N = 81) (HR, 0.51;95% CI 0.30-0.87, p = 30.009). Similar results were found adopting the >26 repeats/<= 26 repeats cut off: (HR, 0.52;95% CI 0.27-0.98, p = 0.035). These data were partially confirmed in the exploratory cohort 2 (TRIBE B): a better median PFS was observed in patients with >26 repeats vs repeats/<= 2626 repeats (N = 205) patients. However, these data were not confirmed in the two validation cohorts. Conclusion: We failed to replicate the exploratory findings in both validation sets. The CCTTT polymorphic region of the INOS gene does not predict outcome in mCRC receiving bevacizumab based first line chemotherapy. Further investigations are needed to reveal mechanisms between tumor, immune system and chemotherapy response.