Abstract Study Objectives To explore the effect of sleep regularity on sleep complaints and mental health conditions (i.e., insomnia, fatigue, anxiety, and depressive symptoms) in a population-based interventional study using a smartphone-based virtual agent. Methods A populational cohort based on the KANOPEE application, which provided interactions with a virtual companion to collect data on sleep and make personalized recommendations to improve sleep over 17 days. A pre-intervention sleep diary and interview were used for cross-sectional analysis (n = 2142), and a post-intervention sleep diary and interview were used for longitudinal analysis (n = 732). The intraindividual mean (IIM) and standard deviation (ISD) of total sleep time (TST) were calculated to measure sleep quantity and sleep regularity. Results The mean age at baseline was 49 years, 65% were female, 72% reported insomnia, 58% fatigue, 36% anxiety and 17% depressive symptoms. Before the intervention, irregular and short sleep was associated with a higher likelihood of insomnia (RR=1.26 [1.21-1.30] for irregular TST and RR=1.19 [1.15-1.23] for short TST), fatigue, anxiety, and depressive symptoms. After the intervention, the IIM of the TST increased while the ISD of the TST and sleep complaints and mental health conditions decreased. More regular TST was associated with reduced insomnia and depressive symptoms (RR=1.33 [1.10-1.52] and RR=1.55 [1.13-1.98], respectively). Conclusions Our results reveal a longitudinal association between sleep regularity and sleep complaints and mental health conditions. Policymakers, health professionals, and the general population should be aware that, beyond its positive effect on sleep health, regular sleep could promote mental health.