Objective To analyze the mutation status of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), v-raf murine sarcoma viral oncogene homolog B1 (BRAF) genes, and the expression of mismatch repair (MMR) and human epidermal growth factor receptor 2 (HER-2) proteins in tumor tissues of patients with colorectal cancer (CRC) harboring neuroblastoma rat sarcoma viral oncogene homolog (NRAS) gene mutations, and explore their relationships with the clinicopathological characteristics of CRC patients. Methods The clinicopathological data of 546 patients with NRAS mutation CRC were retrospectively analyzed. The mutation status of NRAS, KRAS, PIK3CA, and BRAF genes was detected by AmoyDx amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR) kit (fluorescent PCR method), the expression levels of MMR and HER-2 proteins were detected by immunohistochemical staining EnVision method, and the relationship between them and the clinicopathological characteristics of patients were analyzed. Results The mutation rate of single-point mutations in the NRAS gene was 98.35% (537/546), double-point mutations in the NRAS gene were 1.65% (9/546), and double mutations in the NRAS and KRAS genes were 1.47% (8/546). No patients were found to harbor mutations in the PIK3CA or BRAF genes. The types of NRAS mutations included Q61R (or Q61K, Q61L, Q61H) mutations (266/546, 48.72%), G12D (or G12S) mutations (154/546, 28.21%), G13R (or G12C, G12V, G12A, G13V) mutations (134/546, 24.54%), and A146T mutation (1/546, 0.18%). G13R (or G12C, G12V, G12A, G13V) mutations in the NRAS gene were more likely to occur in the rectum cancer patients (P＝0.035); although the tumors had a larger diameter (P＝0.029), the patients had a longer progression-free survival after surgery (P＝0.028). Among patients with NRAS gene mutations, HER-2 positive expression was associated with perineural invasion (P＝0.003), and the patients with deficient MMR were younger on average (P＝0.041) and were associated with double-point mutations in the NRAS gene (P＝0.018). Conclusion CRC harboring NRAS mutations may have unique clinicopathological characteristics and molecular phenotypes, providing possibilities for individualized treatment and prognosis evaluation of CRC.