Our previous studies have shown that camptothecin and doxorubicin triggered ceramide accumulation via de novo synthesis pathway. De novo ceramide generation was responsible for the drug-induced apoptosis through a caspase-3-dependent pathway and a decrease of thrombospondin-1 expression in human thyroid carcinoma FTC-133 cells. Here, we demonstrate that Jun N-terminal kinases play a critical role in camptothecin- and doxorubicin-induced down-regulation of thrombospondin-1 expression: i) de novo ceramide synthesis pathway activates Jun N-terminal kinase 1/2 resulting in activating transcription factor 2 phosphorylation; ii) cell treatment by SP600125, a Jun N-terminal kinase specific inhibitor, strongly reduced activating transcription factor 2 phosphorylation and completely abolished camptothecin and doxorubicin effects; and iii) activating transcription factor 2 expression silencing greatly attenuated camptothecin- and doxorubicin-induced down-regulation of thrombospondin-1 expression and apoptosis. The set of our data established that camptothecin- and doxorubicin-induced activation of Jun N-terminal kinase/activating transcription factor 2 pathway via de novo ceramide synthesis down-regulates thrombospondin-1 expression and apoptosis in human thyroid carcinoma FTC-133 cells.