Early-life adversity is a potent risk factor for mental-health disorders in exposed individuals, and effects of adversity are exhibited across generations. Such adversities are also associated with poor gastrointestinal outcomes. In addition, emerging evidence suggests that microbiota-gut-brain interactions may mediate the effects of early-life stress on psychological dysfunction. In the present study, we administered an early-life stressor (i.e., maternal separation) to infant male rats, and we investigated the effects of this stressor on conditioned aversive reactions in the rats’ subsequent infant male offspring. We demonstrated, for the first time, longer-lasting aversive associations and greater relapse after extinction in the offspring (F1 generation) of rats exposed to maternal separation (F0 generation), compared with the offspring of rats not exposed to maternal separation. These generational effects were reversed by probiotic supplementation, which was effective as both an active treatment when administered to infant F1 rats and as a prophylactic when administered to F0 fathers before conception (i.e., in fathers’ infancy). These findings have high clinical relevance in the identification of early-emerging putative risk phenotypes across generations and of potential therapies to ameliorate such generational effects.