CRE-driven luciferase reporter is commonly used in drug screening systems involving G protein-coupled receptors (GPCRs). In a screen campaign designed to search for melanocortin-4 receptor (MC4R) agonists, podophyllotoxin, a microtubules disruptor, was found to induce cAMP-responsive element (CRE)-driven reporter expression. MC4R was not involved because podophyllotoxin induced CREB activation and CRE-driven transcription in cells not expressing MC4R. Previous studies indicated that intracellular calcium, PKA, and MAPKs are involved in CREB phosphorylation and activation. Our studies revealed that podophyllotoxin did not affect intracellular calcium level and the phosphorylation state of p38. Podophyllotoxin induced JNK and ERK activation, but blockade of JNK and ERK activation with specific inhibitors had no effect on podophyllotoxin-induced CREB activation and CRE-regulated gene expression. Further experiments revealed that H89, a specific inhibitor of PKA, significantly inhibited podophyllotoxin-induced CREB activation. Podophyllotoxin itself did not alter intracellular cAMP level. Taken together, podophyllotoxin induces CREB activation and CRE-driven gene expression via PKA activation by a cAMP-independent mechanism.