The wolf spider Lycosa singoriensis is a hunting spider with a widespread distribution in northwest China. The venom gland of spiders, which is a very specialized secretory tissue, can secrete abundant and complex toxin components. To extensively examine the transcripts expressed in the venom glands of L. singoriensis, we generated 833 expressed sequence tags (ESTs) from a directional cDNA library. Toxin-like sequences account for 69.1% of these ESTs, 17.3% are similar to cellular transcripts and 13.6% have no significant similarity to any known sequences. Here, we identified 223 novel toxin-like sequences, which can be classified into six different superfamilies; that means a novel potential source of ligands for varied ion channels was discovered. With the aid of Gene Ontology terms and homology to eukaryotic orthologous groups, the annotation of cellular transcripts revealed some cellular processes important for the toxin secretion of venom glands including protein synthesis, protein folding, tuned post-translational processing and trafficking, etc.