Background: Neurogenic locus notch homology 4 (Notch-4) is crucial in maintaining stem cells. Special AT-rich sequence-binding protein 2 (SATB-2) is a transcription factor that binds to the nuclear matrix and serves various functions, including brain development. Glutaminase-1 (GLS-1) plays a pivotal role in cancer cell metabolism, growth, and proliferation. This study aims to assess the expression of these markers in colorectal cancer, establishing correlations with clinicopathological characteristics and patient survival. Method: In this retrospective study, we retrieved and analyzed 68 formalin-fixed, paraffin-embedded blocks of primary colorectal adenocarcinoma, not otherwise specified cases, and adjacent normal mucosa. Notch-4, SATB-2 and GLS-1 expressions were analyzed using immunohistochemistry at the Zagazig School of Medicine, Egypt. Results: High expressions of Notch-4 and GLS-1 and low expression of SATB-2 were observed in colonic adenocarcinoma but not in adjacent non-neoplastic mucosa (P < 0.001). High expressions of Notch-4 and GLS-1, along with low expression of SATB-2, were associated with a higher tumor grade, advanced stage (P < 0.001), lymphovascular invasion, lymph node metastasis, and poor disease-free survival and overall survival rates (P < 0.001). Conclusion: High expression of Notch-4 and GLS-1 is correlated with a poor prognosis in colorectal cancer, while high expression of SATB-2 is associated with a favorable prognosis for colorectal carcinoma. These markers can aid in predicting tumor prognosis and guiding targeted therapy for colorectal carcinoma