Previous study indicated that beta1,4-galactosyltransferase I (beta1,4GT1) was up-regulated by cycloheximide (CHX) and thus enhanced apoptosis induced by CHX in SMMC-7721 cells. In this study, we reported that constitutively active Akt protein (myr-Akt) inhibited CHX-induced apoptosis in SMMC-7721 cells through down-regulating beta1,4GT1. However, the two PI3K inhibitors LY294002 and wortmannin treatment up-regulated beta1,4GT1 through enhancing Sp1 protein expression and consequently increased CHX-induced SMMC-7721 cells apoptosis. Besides, our results suggested that beta1,4GT1 and cell surface galactose residues synthesized by elevated beta1,4GT1 played an important role in SMMC-7721 cells apoptosis treated with CHX and PI3K inhibitor together. Moreover, we found that CHX accentuated beta1,4GT1 through down-regulating Akt expression to mediate SMMC-7721 cells apoptosis. Taken together, PI3K inhibitors LY294002 and wortmannin up-regulated beta1,4GT1 and enhanced CHX-induced apoptosis in SMMC-7721 cells, which suggested that PI3K inhibitors might have therapeutic potential when combined with CHX in the treatment of hepatoma.