Neonicotinoid pesticides (NNs) transfer rapidly from mother to offspring, which exhibit neurobehavioral effects. However, no studies have investigated NNs’ transgenerational effects. We exposed F0 generation mice (mothers) to a no-observed-adverse-effect level (NOAEL) of clothianidin (CLO) during gestation and lactation, and examined the adult neurobehavioral effects of three generations of offspring (F1, F2, F3). F1 had lower birth weight, decreased locomotor activity, and increased anxiety-like behavior. In F2, body weight was affected, and there was a decreasing trend in locomotor activity and an increasing trend in anxiety-like behavior. In F3, locomotor activity tended to increase. Thus, even when only the mothers were exposed, the effects of CLOs were still observed in F1, F2, and F3 but the effects became smaller.