Earthworms usually live in the soil, but some earthworms could be found lying dead on the surface of the ground after a heavy rain. UV radiation was suspected to affect earthworm crawling. Since earthworms, Amynthas gracilis, are often observed lying on the soil surface after a heavy rain in Taiwan, the earthworms were used as an animal model in this experiment. Under UV-B exposure, they showed the abnormal behaviors, the S-shape movement and the jumping behavior. Therefore, the purpose of this experiment is to determine how the UV-B radiation affects the earthworm movement behaviors. Some Fos-labelled cells in earthworm ventral nerve cord were shown after the UV-B radiation. This suggested that some neurons were activated by UV-B radiation. The UV-B radiated nerves were found to have higher protein kinase Cα (PKCα) activity, an effect not mediated by direct UV-B absorption. PKC might regulate the calcium channel(s) to increase the intracellular calcium concentration to alter acetylcholine (ACh) release and choline acetyltransferase to affect ACh synthesis in presynaptic cells. The treatment of PKC activator, PMA and calcium ionophore, A23187 both enhanced the abnormal behavior caused by UV-B radiation. And calcium channels blocker, verapamil blocked the jumping behavior caused by UV-B radiation. On the other hand, the UV-B radiation also affected the activity of acetylcholinesterase (AChE), a key enzyme in the synaptic cleft for ACh hydrolysis. Again, AChE activity was not affected by direct UV-B absorption. And AChE inhibitor, chlorpyrifos, treatment enhanced the earthworm behavior. Together, all of those results support that PKCα and AChE are involved in the regulation of earthworm movement caused by UV-B radiation.