Abstract: The bioactivities and proteinous compositions of venoms collected by two different methods from Latrodectus tredecimguttatus were analyzed and compared. Most of proteinous venom components were high-molecular-mass acidic proteins (>10⁴). Compared with the venom obtained from dissected venom gland (DGV), the venom obtained by electrical stimulation (ESV) contained more high molecular mass proteins, but the venom proteins and peptides with low molecular mass (<10⁴) in the preparations were very similar. Intraperitoneal injection of the two venoms in mice gave rise to similar poisoning symptoms and the LD₅₀ values of ESV and DGV were (0.16±0.03) mg/kg and (0.39±0.05) mg/kg, respectively. LD₅₀ values of ESV and DGV in cockroaches (Periplaneta Americana) were 1.87 μg/g and 2.32 μg/g, respectively. ESV was able to block neuromuscular transmission in the mouse phrenic nerve-hemidiaphragm preparation within (25.0±2.2) min at a concentration of 3.2×10^-6g/mL, whereas DGV blocked neuromuscular transmission within (45.7±1.8 )min at the same concentration. The fraction of the venom containing proteins and peptides with molecular mass <10⁴ did not obviously affect the transmission. Those results demonstrated that Latrodectus tredecimguttatus venom was a mixture rich in larger proteins rather than smaller proteins and peptides; the mammalian toxicities of the venom were due primarily to the high molecular weight acidic proteins rather than low molecular weight peptides; the active components contained in ESV and DGV were similar but the content in ESV was higher than that in DGV.