Infant temperament characteristics play a critical role in children’s developmental pathways and can predict adulthood psychopathology. The diversity and composition of the gut microbiota are associated with human temperament in both adults and young children. However, the relationship between the gut microbiota and temperament in 12-month-old infants is rarely studied; this developmental period is when temperament reaches a relatively stable stage. We used high-throughput sequencing methods to explore whether temperament characteristics were associated with gut microbiota diversity and composition. Infants’ fecal samples were collected at 12 months of age for the gut microbiota analysis. Based on the primary caregivers’ reports, infants’ temperaments were measured using the Infant Behavior Questionnaire-revised (IBQ-R). This study included 51 infants, including 20 boys and 31 girls, with a mean age of 12.25 months. Results showed that soothability was positively correlated with maternal education level (β = 0.29, p = 0.043, adjust p = 0.025) and the abundance of Bifidobacterium genera (β = 0.62, p = 0.004, adjust p = 0.002). Conversely, cuddliness was negatively correlated with the abundance of Hungatella genera. There was no significant difference in temperament based on gender. This study demonstrated that gut microbiota composition was associated with temperament in 12-month-old infants. These results point to the importance of gut microbiota balance. Future studies on the mechanisms behind the gut microbiota affecting temperament are warranted.