Here, we demonstrate that time-of-day determines the efficacy of NAD+ treatment for diet-induced metabolic disease in mice. Increasing NAD+ prior to the active phase in obese male mice ameliorated metabolic markers including body weight, glucose and insulin tolerance, hepatic inflammation and nutrient sensing pathways. However, raising NAD+ immediately before the rest phase selectively compromised these responses....In humans, clinical trials aiming to boost endogenous NAD+ for treatment of metabolic diseases are increasing, showing promising results... However, all these studies mostly overlook the time of drug intake, which is selected based on practicalities or attempting to displace side effects from the patient’s active phase. Considering our results, we propose that time of treatment dictates the amplitude of metabolic benefits from rising NAD+ levels, which ideally outlines the basic strategy of chronobiology-based NAD+ therapy.