Recurrent airway obstruction (RAO) is a chronic inflammatory airway disease affecting mature horses exposed to airborne substances and characterized by neutrophil influx into airways, reminiscent of induced asthma in humans. Asthmatic and RAO-affected individuals are deficient in the epithelial cells that secrete secretoglobin (SCGB) 1A1, an anti-inflammatory protein, into airway surface fluids. Herein, we characterized three equine SCGB1A1 genes and measured their transcripts in tissues using PCR assays. SCGB1A1 genes differed from each other by ~10 nucleotides, and coded for different proteins. Transcripts were detected for SCGB1A1 and SCGB1A1A, but not for SCGB1A1P. Bioinformatic analysis did not identify elements in the proximal promoter region to mediate silencing of SCGB1A1P. Among 33 tissues evaluated, SCGB transcripts were most abundant in lung, uterus, Fallopian tube and mammary gland, which correlated with tissue detection of SCGB proteins by immunohistochemistry. Investigation of SCGB1A1 genes in other equid species identified three genes in E. przewalskii, two genes in E. asinus, and a single gene in E. grevyi and E. quagga, indicating that the evolution of SCGB1A1 likely paralleled diversification within the Equidae family. Most of the non-synonymous nucleotide substitutions between the different equid genes localized to the SCGB central cavity that binds hydrophobic ligands, implying that SCGB genes evolved to accommodate diverse molecular interactions. Recombinant equine SCGB 1A1 and 1A1A were produced, and assessment of their effect on neutrophil function showed that SCGB 1A1A but not 1A1 increased neutrophil oxidative burst and phagocytosis, whereas both proteins markedly reduce neutrophil chemotaxis and neutrophil extracellular trap (NET) formation. NETs were also detected in bronchoalveolar lavage fluid from horses with exacerbated RAO but not in horses with RAO in remission or in healthy horses exposed to inhaled challenge material. In summary, the studies identified the first SCGB1A1 gene triplication in a mammalian genome and showed that the SCGB1A1 locus evolved and produced two distinct but functional equine genes. The genes are differentially expressed in the lung of horses with RAO, and the proteins have unique effects on neutrophil function. In vivo significance of the different proteins remains to be fully explored.