Importance: Infantile hemangiomas (IHs) vary substantially in localization and extent of tissue involvement, but IH biological progression is remarkably unique and predictable. Propranolol is an effective treatment for symptomatic IH, but its mechanism of action remains unknown and understudied., Objective: To compare excreted proteins in infants with IH being treated with propranolol vs prednisolone., Design, Setting, and Participants: Exploratory urine proteomics profiling of patients with IH from July 2010 to September 2012 at a tertiary pediatric hospital. Participants were infants with IH treated at our institution who were participating in a blinded, randomized trial comparing prednisolone vs propranolol. They ranged in age from 14 days to 15 months at enrollment. Exclusion criteria included a history of diabetes mellitus, asthma, and/or cardiovascular disease including hypertension or hypotension. Urine samples were longitudinally collected from all participants. Specimens were desalted, concentrated, and gel fractionated, and the protein content was identified using liquid chromatography tandem mass spectrometry. Western blot analyses and enzyme-linked immunosorbent assays (ELISAs) were performed to validate mass spectrometry findings., Intervention: Treatment with propranolol or prednisolone administered starting before the age of 6 months., Main Outcomes and Measures: Proteins present in urine samples and change in urinary levels of proteins over time., Results: Samples were obtained from 3 patients treated with prednisolone, 3 patients treated with propranolol, and 5 untreated controls with IH. More than 1000 urinary proteins were identified by proteomics. Patients treated with propranolol demonstrated attenuation of excreted matrix metalloproteinase 9 (MMP-9) in urine over the proliferative phase of the condition compared with prednisolone-treated patients. These findings were validated with Western blot analysis and quantified with ELISA, which confirmed mean urinary MMP-9 levels in the first year of life to be significantly lower in propranolol-treated patients with IH compared with prednisolone-treated patients with IH (0.118 vs 0.501 ng/mL; P = .03) or with nontreated patients with IH (0.118 vs 3.69 ng/mL; P = .02)., Conclusions and Relevance: Propranolol treatment decreases urinary excretion of MMP-9 in patients with IH. Matrix metalloproteinase 9 may be a biomarker for IH propranolol responsiveness, and its signaling pathways may represent the molecular target of this drug.