Background: Human being needs sunlight for physical and mental well-being. Sunlight helps the body to produce vitamin D, an important vitamin for skeletal development, immune function, and blood cell formation. So, the community should be counseled to get sufficient sun exposure and vitamin D supplementation to uphold the serum 25 (OH) D levels. This study designed to assess the mothers’ knowledge, practice, and factors affecting the sunlight exposure of their infants. Methods: A facility-based cross-sectional study was conducted at governmental health facilities in Dessie Town, 2021. A total of 398 mothers were interviewed using semi structured questionnaires. Data were entered into the EPI data version 3.1 and analyzed using SPSS 23. Binary and multivariate logistic regression analyses were also performed. In multivariate analysis, a significant association was considered at P-value of <.05. Results: Response rate was 98.9%. About 76.6% and 58.9% of the mothers had poor knowledge and poor practice regarding sunlight exposure of infants respectively. Mothers’ occupation (aOR = 0.124, 95% CI = 0.042, 0.365), mothers’ source of information (aOR = 18.604, 95% CI = 7.564, 45.75), and attitude (aOR = 2.773, 95% CI = 1.474, 5.215) had showed a significant association with mothers’ knowledge. On the other hand, mothers’ age (aOR = 3.191, 95% CI = 1.334, 7.633), mothers occupation (aOR = 4.226, 95% CI = 2.321, 7.694), and baby age (aOR = 1.989, 95% CI = 1.260, 3.140) had a significant association with their practice about sunlight exposure of infants. Conclusion: Mothers’ knowledge and practice of sunlight exposure of infants are poor in Dessie Town. Hence measures should be taken to increase and improve mothers’ responsiveness of sunlight exposure of infants.