STUDY OBJECTIVES: Alterations in gut microbiota composition have been associated with several conditions, and there is emerging evidence that sleep quantity and quality are associated with the composition of the gut microbiome. Therefore, this study aimed to assess the associations between several measures of sleep and the gut microbiome in a large, population-based sample. METHODS: Data were collected from participants in the Survey of the Health of Wisconsin from 2016 to 2017 (N = 720). Alpha diversity was estimated using Chao1 richness, Shannon's diversity, and Inverse Simpson's diversity. Beta diversity was estimated using Bray-Curtis dissimilarity. Models for each of the alpha-diversity outcomes were calculated using linear mixed effects models. Permutational multivariate analysis of variance tests were performed to test whether gut microbiome composition differed by sleep measures. Negative binomial models were used to assess whether sleep measures were associated with individual taxa relative abundance. RESULTS: Participants were a mean (SD) age of 55 (16) years and 58% were female. The sample was 83% non-Hispanic white, 10.6% non-Hispanic black, and 3.5% Hispanic. Greater actigraphy-measured night-to-night sleep duration variability, wake-after-sleep onset, lower sleep efficiency, and worse self-reported sleep quality were associated with lower microbiome richness and diversity. Sleep variables were associated with beta-diversity, including actigraphy-measured night-to-night sleep duration variability, sleep latency and efficiency, and self-reported sleep quality, sleep apnea, and napping. Relative abundance of several taxa was associated with night-to-night sleep duration variability, average sleep latency and sleep efficiency, and sleep quality. CONCLUSIONS: This study suggests that sleep may be associated with the composition of the gut microbiome. These results contribute to the body of evidence that modifiable health habits can influence the human gut microbiome.