Interleukin (IL)-8 plays the critical role in the initiation of micro-environmental inflammation responsible for tumour growth and patient prognosis. This study aimed at investigating the molecular mechanisms of IL-8 production from human hepatocellular carcinoma (HCC) cells. The levels of IL-8 and phosphorylation of p38 mitogen-activated protein kinase (MAPK), ERK1/2 and Akt in MHCC-97H cells were measured by ELISA, Western blot and immunofluorescence. NF-κB p65 protein nuclear translocation was determined by non-radioactive NF-κB p50/p65 transcription factor activity kit and cell bio-behaviours were detected by the real-time cell-monitoring system. Tumour necrosis factor-α (TNF-α) significantly induced phosphorylation of p38 MAPK, ERK, Akt and production of IL-8 from HCC cells, which were prevented by SB203580 (p38 MAPK inhibitor), PD98059 (ERK inhibitor), LY294002 and Wortmannin (PI3K inhibitor) and SB328437 (CCR3 inhibitor). TNF-α could significantly increase the translocation of NF-κB p65 protein into the nucleus in a dose-dependent manner, while SB203580 partially inhibited. In inflammatory micro-environment, HCC auto-produced IL-8 through p38 MAPK, ERK and PI3K/Akt signalling pathways, where the p38 MAPK is a central factor to activate the NF-κB pathway and regulate the expression of IL-8 production. There was a potential cross-talking between receptors.