Toxicological studies have shown that the cockroach Periplaneta americana is a classical model for studying the mode of action of commonly used insecticides. In a previous study, we demonstrated that thiamethoxam and clothianidin decreased locomotor activity and decreased the locomotor activity in an open-field-like apparatus. Here, we tested the effect of the neonicotinoid acetamiprid when applied orally, topically, or injected into the haemolymph. We found that acetamiprid decreased locomotor activity in the open-field-like apparatus. When treated with acetamiprid, a strong alteration in locomotor activity was observed 1 h, 24 h and 48 h after haemolymph and topical applications. Oral application of acetamiprid demonstrated that it impaired locomotor activity at 24 h and 48 h. A comparison of the present data with our previously published results showed that neonicotinoids were more active when injected into the haemolymph compared to oral and topical applications. These findings increased our understanding of the effect of neonicotinoid insecticides on insect locomotor activity, and demonstrated that the cyano-substituted neonicotinoid, acetamiprid, was able to alter cockroach locomotor activity.