Background: Noise annoyance is associated with adverse health-related conditions and reduced wellbeing. Thereby, subjective noise annoyance depends on the objective noise exposure and is modified by personal and regional factors. Objective: How many participants of the German National Cohort Study (GNC; NAKO Gesundheitsstudie) were annoyed by transportation noise during nighttime and what factors were associated with noise annoyance? Materials and methods: This cross-sectional analysis included 86,080 participants from 18 study centers, examined from 2014 to 2017. We used multinomial logistic regression to investigate associations of personal and regional factors to noise annoyance (slightly/moderately or strongly/extremely annoyed vs. not annoyed) mutually adjusting for all factors in the model. Results: Two thirds of participants were not annoyed by transportation noise during nighttime and one in ten reported strong/extreme annoyance with highest percentages for the study centers Berlin-Mitte and Leipzig. The strongest associations were seen for factors related to the individual housing situation like the bedroom being positioned towards a major road (OR of being slightly/moderately annoyed: 4.26 [95% CI: 4.01;4.52]; OR of being strongly/extremely annoyed: 13.36 [95% CI: 12.47;14.32]) compared to a garden/inner courtyard. Participants aged 40–60 years and those in low- and medium-income groups reported greater noise annoyance compared to younger or older ones and those in the high-income group. Conclusion: In this study from Germany, transportation noise annoyance during nighttime varied by personal and regional factors.