Background: Mutations in the EPCAM gene can lead to the absence of this protein from epithelial cell surfaces, resulting in various disorders such as Lynch syndrome, congenital tufting enteropathy, cholestatic liver disease, and a range of cancers. Therefore, analyzing mutations in this gene is significant from diagnostic and prognostic perspectives. This study examines the effect of various mutations in the EPCAM gene on different attributes of the encoded protein. Objective: The objective of this study was to analyze the impact of single nucleotide polymorphisms (SNPs) in the EPCAM gene on the protein's structural and functional properties, aiming to identify potential predictive biomarkers for EPCAM-associated diseases. Methods: The study focused on the EPCAM gene transcript EPCAM-201, with ENSEMBL transcript ID ENST00000263735.9 and NCBI Reference Sequence NM_002354.3. The gene sequence was retrieved from the NCBI database, and SNPs were selected from the ENSEMBL database. A total of 21 variants were selected to design twenty-one cases. These cases were analyzed using various bioinformatics tools, including EXPASY for nucleotide sequence translation, HOPE server for 3D structure analysis, CELLO2GO for sub-cellular localization, and PROTPARAM for physicochemical parameter prediction. Data collection adhered to the principles outlined in the Declaration of Helsinki. Statistical analyses were conducted using SPSS version 25, with descriptive statistics and appropriate tests to determine the significance of the observed variations. Results: Analysis revealed that sixteen SNPs, namely rs994384264, rs1280024892, rs1294456118, rs149875996, rs767811939, rs750826481, rs754293486, rs748292053, rs1460762372, rs1294456118, rs149875996, rs754293486, rs748292053, rs771063031, rs776854951, and rs267606785, significantly altered the 3D structure of mutated proteins. Among these SNPs, rs994384264 and rs149875996 also affected the sub-cellular localization of proteins. Additionally, four mutations—rs1280024892, rs1460762372, rs987919056, and rs771029207—caused alterations in extinction coefficient, isoelectric point (pI), aliphatic index, and instability index. Conclusion: These single nucleotide variants might serve as predictive biomarkers for EPCAM-associated diseases, aiding in diagnosis and prognosis. Variants predicted in this study require further experimental validation to confirm their clinical utility.