Abstract: Objective: To explore the molecular mechanism by which cancer-testis antigen 23 (CT23) participates in the regulation of the pentose phosphate pathway (PPP) and promotes apoptosis in hepatocellular carcinoma (HCC) cells. Methods: The relationship between CT23 and PPP was investigated by whole transcriptomic sequencing, glucose consumption detection, lactic acid production analysis, reduced nicotinamide adenine dinucleotide phosphate (NADPH) production detection, reactive oxygen species (ROS) production analysis and mitochondrial tracer analysis.Western blotting and reverse transcription-quantitative PCR (RT-qPCR) were used to detect the expression of hexose-6-phosphate dehydrogenase (H6PD) in HCC cells with CT23 knockdown, and the apoptosis was analyzed by the TUNEL assay. Results: CT23 was related to PPP.Compared with the control group, HCC cells in the shCT23 group had decreased glucose consumption, decreased lactate production level, decreased NADPH production level, increased ROS level, increased apoptosis, decreased H6PD mRNA level and H6PD protein level (all P< 0.05).The morphological changes of the cells were accompanied by mitochondrial damage under electron microscope.Compared with the shCT23 group, HCC cells in shCT23+H6PD^OE group had increased H6PD protein level, increased glucose consumption, increased lactate production level, increased NADPH production, and decreased apoptosis (all P< 0.05). Conclusion: CT23 promotes HCC cell apoptosis through enhancing PPP via H6PD.