Venomous animals use venom, a complex biofluid composed of unique mixtures of proteins and peptides, for either predation or defense. Bumblebees, which occur in various habitats due to their unique thermoregulatory properties, mainly use venom for defense. Herein, we conducted an exploratory analysis of the venom composition of a bumblebee species (Bombus pascuorum) along an elevation gradient in the western Swiss Alps using shot-gun proteomic approaches to assess whether their defense mechanism varies along the gradient. The gradient was characterized by high temperatures and low humidity at low elevations and low temperatures and high humidity at high elevations. Venom composition is changing along the elevation gradient, with proteomic variation in the abundances of pain-inducing and allergenic proteins. In particular, the abundance of phospholipase A ₂ -like, the main component of bumblebee venom, gradually decreases toward higher elevation (lower temperature), suggesting venom alteration and thus a decrease in bumblebee defense towards harsher environments. Larger datasets may complement this study to validate the observed novel trends.