Background Betanin, a natural pigment and glycosidic bioactive compound, possesses anti-inflammatory antioxidant activity. Objectives In this study, we evaluated the potential effect of betanin on cigarette smoke extract (CSE)-induced pulmonary inflammation in the zebrafish model. Materials and Methods Zebrafish were randomly divided into five groups with control, CSE-exposed and betanin-treated after CSE exposure for 3 and 6 days. Oxidative stress-related parameters like reactive oxygen species (ROS), nitric oxide (NO) and myeloperoxidase (MPO) were analysed, histopathological studies were carried out and gene expression of proinflammatory tumour necrosis factor-alpha, inducible NO synthase and anti-inflammatory interleukin-10 cytokines was determined. Results Treatment with betanin reduced ROS, MPO and NO with histopathological improvement, alleviating CSE-induced pulmonary inflammation. Further, decreased expression of the proinflammatory gene and increased expression of the anti-inflammatory gene were observed after 6 days of treatment. Conclusion Betanin exhibits a protective effect against CSE exposure by inhibiting oxidative stress and inflammation.