Arsenic trioxide (ATO) has shown anticancer activity against a variety of solid tumor models through induction of apoptosis, promotion of cellular differentiation, and inhibition of cellular invasive ability. The present study investigated the role of ceramide in regulating the invasive activity of hepatoma carcinoma HCCLM3 cells during ATO treatment. We found that ATO treatment inhibited HCCLM3 cell invasion and downregulated matrix metalloproteinase-9 (MMP-9) protein levels in a concentration-dependent manner. ATO also dose dependently induced the generation and accumulation of ceramide in HCCLM3 cells. Blockage of intracellular ceramide production through the inhibition of de novo ceramide synthesis or the hydrolysis of sphingomyelin increased the invasive ability and upregulated MMP-9 protein levels. The findings of this study indicated that ATO induced ceramide production through de novo ceramide synthesis and the hydrolysis of sphingomyelin and suggested that ceramide accumulation in response to ATO stimuli may play an important role in cancer therapy.