Cockroach neurosecretory cells, dorsal unpaired median (DUM) neurons, express two distinct α-bungarotoxin-insensitive nicotinic acetylcholine receptor subtypes, nAChR1 and nAChR2 which are differently sensitive to the neonicotinoid insecticides and intracellular calcium pathways. The aim of this study is to determine whether sulfoxaflor acts as an agonist of nAChR1 and nAChR2 subtypes. We demonstrated that 1 mM sulfoxaflor induced high current amplitudes, compared to acetylcholine, suggesting that it was a full agonist of DUM neuron nAChR subtypes. Sulfoxaflor evoked currents were not inhibited by the nicotinic acetylcholine receptor antagonist d-tubocurarine (dTC) which reduced nAChR1. But, sulfoxaflor evoked currents were reduced in the presence of 5 μM mecamylamine which is known to reduce nAChR2 subtype. Interestingly, when 1 μM imidacloprid was added in the extracellular solution, sulfoxaflor-induced currents were significantly suppressed. Moreover, when extracellular calcium concentration was increased, bath application of 1 μM imidacloprid partially reduced sulfoxaflor activated currents when nAChR1 was inhibited with 20 μM dTC and completely suppressed sulfoxaflor currents when nAChR2 was inhibited with 5 μM mecamylamine. Our data demonstrated therefore that sulfoxaflor activates both nAChR1 and nAChR2 subtypes.