Profilins are ubiquitous pan-allergens responsible for cross-reactivity between pollens and plant foods. While we previously demonstrated that recombinant Acanthamoeba profilin (rAc-PF) drives allergic airway inflammation via Th2/Th17 pathways in murine models, its clinical relevance in human allergic disease remains unclear. This study investigated rAc-PF as a novel inhalant allergen and its immunological impact on patients with allergic airway diseases. A total of 176 patients with allergic airway diseases underwent skin prick tests with rAc-PF and a standard panel of 55 common aeroallergens. Acanthamoeba-specific and rAc-PF–specific serum IgE levels were quantified using ELISA. To elucidate immune mechanisms, peripheral blood mononuclear cells from atopic asthma patients were stimulated with rAc-PF, and Th2/Th17 cytokine production was analyzed. Thirteen patients (7.4%) showed positive skin prick tests reactions to rAc-PF. This sensitization was significantly associated with tree, grass, and weed pollens, indicating a pan-allergen characteristic due to high cross-reactivity. Notably, one patient sensitized to rAc-PF reacted to no other common inhalants, suggesting rAc-PF as a unique causative agent. Patients exhibited significantly elevated levels of serum Acanthamoeba-specific and rAc-PF–specific IgE compared to healthy controls. Furthermore, rAc-PF stimulation of peripheral blood mononuclear cells from asthmatic patients induced robust production of IL-4, IL-5, IL-13, and IL-17A in humans. rAc-PF is identified as a novel allergen capable of inducing IgE-mediated sensitization and mixed Th2/Th17 responses. The strong association with pollen sensitization supports its role as an environmental pan-allergen. Therefore, rAc-PF should be considered a clinically relevant diagnostic target, especially in patients with polysensitization or unidentified triggers.