The Brown Treesnake (Boiga irregularis), a rear-fanged member of the polyphyletic family Colubridae, is an introduced predator on Guam which has been responsible for numerous human envenomations. Because little is known about this species’ venom, we characterized venom proteins from B. irregularis using enzyme assays, one and 2D electrophoresis, Western blot analysis, mass spectrometry, HPLC and toxicity assays. Venom yields and protein content varied signiﬁcantly with snake size, and large adult specimens averaged over 500 ml venom (19.2 mg, protein content w90%). Only two enzymes, azocaseinolytic metalloprotease and acetylcholinesterase, were detected in venoms, and both activities increased with snake size/age. Western blot analysis demonstrated a 25 kDa CRiSP homolog in venoms from both neonate and adult snakes. 2D electrophoresis showed variation between venoms from neonate and adult snakes, especially with respect to metalloprotease and acetylcholinesterase. Analysis by MALDI-TOF mass spectrometry revealed the presence of numerous proteins with molecular masses of w8.5–11 kDa. Adult B. irregularis venom was quite toxic to domestic chickens (Gallus domesticus; 1.75 mg/g) and lizards (Hemidactylus geckos: 2.5 mg/g and Carlia skinks: 4.5 mg/g), and intoxication was characterized by rapid paralysis of all species and neck droop in chickens. Toxicity of venom from neonates toward geckos was 1.1 mg/g, consistent with the presence of a greater diversity of 8–11 kDa proteins (suspected neurotoxins) in these venoms. All of these values were notably lower than murine LD50 values (neonate: 18 mg/g; adult: 31 mg/g). Like venoms of several front-fanged species, B. irregularis venom showed an ontogenetic shift in enzyme activities and toxicity, and neonate snakes produced more toxic venoms with lower protease and acetylcholinesterase activities. High toxicity toward non-mammalian prey demonstrated the presence of taxa-speciﬁc effects (and thus toxins) in B. irregularis venom, likely a characteristic of many colubrid snake venoms. We hypothesize that the lack of signiﬁcant envenomation effects in humans f