Influenza A virus (IAV) infection affects the human respiratory tract, causing an acute and highly contagious disease. Individuals with comorbidities and in the extremes of age are classified as risk groups for serious clinical outcomes. However, part of the severe infections and fatalities are observed among young healthy individuals. Noteworthy, influenza infections lack specific prognostic biomarkers that would predict the disease severity. Osteopontin (OPN) has been proposed as a biomarker in a few human malignancies and its differential modulation has been observed during viral infections. However, OPN expression levels in the primary site of IAV infection have not been previously investigated. Therefore, we evaluated the transcriptional expression patterns of total OPN (tOPN) and its splicing isoforms (OPNa, OPNb, OPNc, OPN4, andOPN5) in 176 respiratory secretion samples collected from human influenza A(H1N1)pdm09 cases and a group of 65 IAV-negative controls. IAV samples were differentially classified according to their disease severity.tOPNwas more frequently detected in IAV samples (34.1%) when compared with the negative controls (18.5%) (p< 0.05), as well as in fatal (59.1%) versus non-fatal IAV samples (30.5%) (p< 0.01).OPN4splice variant transcript was more prevalent in IAV cases (78.4%) than in the negative controls (66.1%) (p= 0.05) and in severe cases (85.7%) in relation to the non-severe ones (69.2%) (p< 0.01).OPN4detection was also associated with severity symptoms such as dyspnea (p< 0.05), respiratory failure (p< 0.05), and oxygen saturation < 95% (p< 0.05). In addition, theOPN4expression level was increased in the fatal cases of respiratory samples. Our data indicated thattOPNandOPN4had a more pronounced expression pattern in IAV respiratory samples, pointing to the potential use of these molecules as biomarkers to evaluate disease outcomes.