The sialic acid-specific cytotoxic lectin viscumin and its recombinant equivalent rViscumin specifically bind to CD75s-gangliosides with terminal Neu5Acα6Galβ4GlcNAc sequence. We, therefore, comparatively analyzed the content of CD75s-gangliosides and closely related iso-CD75s-gangliosides (terminated by Neu5Acα3Galβ4GlcNAc sequence) and the gene expression of associated β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) and β-galactoside α-2,3-sialyltransferase 6 (ST3GAL6), respectively, in 35 hepatocellular carcinoma (HCC) patients. Ganglioside structures were identified in lipid extracts of matched pairs of malignant and nonmalignant liver tissues by thin-layer chromatography immunodetection coupled with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. CD75s- and iso-CD75s-gangliosides were found to be deregulated in tumor tissues and showed an elevated occurrence in 35 and 41% of HCCs, respectively, compared with nontumoral liver tissues. Statistical analysis revealed a correlation between enhanced iso-CD75s-ganglioside amount and a poor histopathological differentiation (τ = 0.317, P = 0.045) and a significant association of CD75s- and iso-CD75s-ganglioside levels in nontumorous (τ = 0.392, P = 0.003) and in tumorous tissues (τ = 0.650, P < 0.001). Quantitative real-time polymerase chain reaction gene expression analysis of sialyltransferases exhibited no difference in ST6GAL1 expression in cancerous and adjacent noncancerous tissues. Interestingly, the ST3GAL6 expression was significantly diminished in HCCs (P = 0.003). The results indicate that the occurrence of CD75s- and iso-CD75s-gangliosides in tumor tissues is largely independent of the transcriptional expression of ST6GAL1 and ST3GAL6, respectively. Thus, further experiments are required to explore the rationale behind the differential ganglioside level and to validate the applicability of CD75s- and iso-CD75s-gangliosides as targets for individual HCC therapies.