Cancer testis antigens (CTAs) are an extensive gene family with a unique expression pattern restricted to germ cells, but aberrantly reactivated in cancer tissues. Studies indicate that the expression (or re-expression) of CTAs within the MAGE-A family is common in hepatocellular carcinoma (HCC). However, no systematic characterization has yet been reported. The aim of this study is to perform a comprehensive profile of CTA de-regulation in HCC and experimentally evaluate the role of MAGEA3 as a driver of HCC progression. The transcriptomic analysis of 44 multi-regionally sampled HCCs from 12 patients identified high intra-tumor heterogeneity of CTAs. In addition, a subset of CTAs was significantly overexpressed in histologically poorly differentiated regions. Further analysis of CTAs in larger patient cohorts revealed high CTA expression related to worse overall survival and several other markers of poor prognosis. Functional analysis of MAGEA3 was performed in human HCC cell lines by gene silencing and in a genetic mouse model by overexpression of MAGEA3 in the liver. Knockdown of MAGEA3 decreased cell proliferation, colony formation and increased apoptosis. MAGEA3 overexpression was associated with more aggressive tumors in vivo. In conclusion MAGEA3 enhances tumor progression and should be considered as a novel therapeutic target in HCC. Author summary Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. Several recurrent driver genes of HCC have been established, yet no targeted drug against these drivers has been approved for the treatment of HCC. Novel targets for therapeutic development are desperately needed. This study aims to identify novel drivers of tumor progression from low-grade tumors to high-grade, aggressive malignancies. We collected multiple biopsies of single tumors from 12 patients and performed RNA-sequencing. We hypothesized that we could identify important genes to HCC progression by identifying differentially up-regulated genes in high-grade regions compared to low-grade regions of the same tumor. Interestingly, we found that one family of genes was recurrently over-expressed in the most aggressive regions of the tumors: cancer testis antigens (CTAs). Here we show that X-chromosome located CTAs, and especially MAGEA3, are associated with markers of poor prognosis in HCC. Experimentally, inhibiting expression of MAGEA3 in HCC cell lines decreases the ability for cells to proliferate and causes cells to undergo cell death. In mice, over-expressing MAGEA3 in the liver along with known HCC drivers causes mice to succumb to disease faster. Changes in RNA and protein levels within HCC cells after MAGEA3 inhibition suggests that maintenance of Survivin, an apoptosis inhibiting protein, plays an important role in how MAGEA3 drives HCC progression. Overall, our study identified the CTA MAGEA3 as important for HCC progression and a novel therapeutic target.