Amebiasis, caused by the protozoan , is a significant parasitic infection affecting millions of people worldwide, particularly in developing regions. Elucidating the mechanisms by which this parasite invades host tissues and circumvents immune defenses is essential for advancing therapeutic strategies. Key virulence factors such as Gal/GalNAc lectin, amebapore, and proteases enable the parasite to adhere to, invade, and destroy host tissues. This study aimed to identify novel virulence-related genes in clinical strains by analyzing their gene expression profiles. We collected clinical isolates from asymptomatic individuals and patients with amoebic liver abscesses to perform RNA sequencing and compare their gene expression profiles. The analysis identified 14 differentially expressed genes between high-virulence and low-virulence strains. Among these, four candidate genes exhibited significant upregulation in the virulent strains. Functional assays demonstrated that the overexpression of these genes contributed to key virulence traits, including increased adhesion, complement resistance, and enhanced starch phagocytosis. Significantly, two of the four candidate genes, EHI_124550 and EHI_107170, which encode hypothetical proteins, exhibited a strong correlation with both oxidative stress response and complement resistance. These findings suggest that specific genes play crucial roles in the parasite's ability to evade the host immune system and establish infection in extraintestinal sites like the liver.IMPORTANCEThis study focuses on understanding how , the parasite responsible for amebiasis, affects over 50 million people globally. Our research is the first study to examine various clinical strains of the parasite, identifying key genes that influence its ability to attach to host cells (adhesion) and ingest them (phagocytosis), both critical processes for its ability to cause disease. Additionally, we discovered that these genes play a role in helping the parasite withstand environmental stress, such as oxidative stress and heat shock, which are part of the body's defense mechanisms. These findings are significant because they reveal potential targets for future treatments aimed at reducing the parasite's virulence, or disease-causing potential. Understanding how adapts and survives under hostile conditions will help in developing better strategies to combat amebiasis. These results provide new insights into a unique immune evasion strategy employed by a pathogen.