Constipation is prevalent in individuals after stroke. However, the pathophysiological mechanisms of poststroke constipation remain unclear. This study was designed (i) to investigate the difference in anorectal motility and rectal sensation among stroke patients with constipation, stroke patients without constipation, and healthy controls (HC), (ii) to evaluate the impact of stroke sites on constipation and rectal sensation, (iii) to explore the role of autonomic functions, and (iv) to determine the independent risk factors for poststroke constipation. Seventy-one stroke patients and 24 HC were recruited. General information, clinical characteristics, and relevant questionnaires were collected. Meanwhile, an anorectal manometry test was performed to assess functions of anorectal motility and rectal sensation, and an electrocardiogram was recorded to evaluate autonomic functions. (i) Constipation patients exhibited increased rectal sensation thresholds, compared with patients without constipation or HC (P < 0.001). Almost no difference was detected in anorectal motility parameters among 3 groups. Constipation-associated clinical characteristics, such as spontaneous bowel movements, were weakly or moderately correlated with rectal sensation thresholds (P < 0.05 to P < 0.001 for various parameters). (ii) Patients with brainstem lesions had increased prevalence of constipation and first sensation threshold, compared with patients without brainstem lesions (P = 0.045, P = 0.025, respectively). (iii) There was a weak positive correlation between sympathetic activity and stroke severity and a weak negative one between vagal activity and stroke severity. Rectal sensation thresholds were positively and weakly correlated with sympathetic activity but negatively with vagal activity. (iv) The desire of defecation threshold and the physical activity were independent risk factors for poststroke constipation (P = 0.043, P = 0.025, respectively). Poststroke constipation is characterized by elevated thresholds for rectal sensation, rather than altered anorectal motility. Patients with brainstem lesions are predisposed to constipation possibly because of the disruption of afferent pathway from the rectum to the brain. Moreover, the desire of defecation threshold and the physical activity level are factors independently associated with poststroke constipation.