Daphnetin has been shown to be a potent in vitro anti-proliferative agent to the human renal cell carcinoma (RCC) cell line, A-498. In the present study, we investigated its effects on mitogen-activated protein kinase (MAPK) signalling along with cell cycle events and cellular differentiation. Daphnetin-activated p38, however, higher concentrations were required to inhibit ERK1/ERK2. In addition, it did not activate SAPK or induce apoptosis, but instead inhibited S phase cell cycle transition of A-498 cells at low concentrations and time of exposure. In addition, a late G(1), early S phase inhibition was observed at higher concentrations and time of exposure, indicating that the mechanism of daphnetin-induced differentiation was concentration dependent. Increased expression of the epithelial differentiation markers cytokeratins 8 and 18, correlated with increasing concentrations of daphnetin, while pre-treatment with a specific p38-inhibitor, served to limit this effect. There was no evidence that P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) played a role in the anti-proliferative activity of daphnetin. Consequently, we concluded that p38 MAP kinase is intrinsically involved in mediating the effect of daphnetin in A-498 cells, suggesting that this drug may act by promotion of cellular maturation, and consequently may represent a novel low toxic approach for the treatment of poorly differentiated RCCs.