Objective To investigate the effect of osteopontin (OPN) interference on the Warburg effect in hepatoma cells and to explore the related molecular mechanism. Methods Small interfering ( si) RNA targeting OPN (si-OPN) and negative Control siRNA (si-NC) were transfected into HepG2 and SK-HEP-1 cells. The glucoseuptake capacity of hepatoma cells was detected using a fluorescent probe ( 2-NBDG), and lactate production in HepG2 and SK-HEP-1 cells was evaluated using a lactate-detection kit. Expression levels of the glycolysis-related genes and proteins glucose transporter type 1 ( GLUT1), hexokinase 2 ( HK2), and lactate dehydrogenase A (LDHA) were determined by quantitative reverse transcription-polymerase chain reaction and Western blot. We then silenced the expression of galectin-3-binding protein (LGALS3BP) in HepG2 and SK-HEP-1 cells, and overexpressed LGALS3BP in OPN-silenced HepG2 and SK-HEP-1 cells, and evaluated the expression levels of LGALS3BP,GLUT1, HK2, and LDHA, and the glucose-uptake capacity and lactate production in the above cells. ResultssiOPN significantly reduced glucose uptake and lactate production in HepG2 and SK-HEP-1 cells compared with the Control and si-NC groups, and significantly reduced GLUT1, HK2, and LDHA gene and protein expression levels (P<0. 05). si-OPN reduced LGALS3BP protein expression in HepG2 and SK-HEP-1 cells, and silencing LGALS3BP reduced glucose uptake, lactate generation, and the expression levels of GLUT1, HK2, and LDHA ( P<0. 05).Overexpression of LGALS3BP rescued the suppression of the above glycolytic metabolism-related indicators ( P<0. 05). These result suggest that OPN stimulated glycolytic metabolism in hepatocellular carcinoma cells via LGALS3BP. Conclusions OPN can affect the expression of glycolysis-related genes via LGALS3BP, thereby regulating the Warburg effect in hepatoma cells.