The tarantula Chilobrachys jingzhao is one of the most venomous spiders with a specialized organ, venom gland, which synthesizes and secretes the complex and abundant toxin proteins. The components of the venom have been extensively studied. As far as the molecular mechanism of toxin secretion and metabolism is concerned, we still knew a little. To obtain a comprehensive view of function of its venom gland we constructed a non-normalized cDNA library of the venom gland and generated 788 expressed sequence tags (ESTs). All ESTs were assembled into 356 non-redundancy sequences including 85 clusters and 271 singlets, of which 31.4% of total unique sequences belong to secretion protein coding sequences including cystine knot toxins (29.1%) and other secretion proteins (2.3%); 54.0% are similar to common cellular transcripts; and 14.6% have no significant similarity to any known sequences. Annotation of these ESTs revealed some novel possible venom components and cellular processes important for venom gland functions, including protein posttranslation processing, cell motility, protein synthesis, energy supply, etc. This study contains a transcriptome analysis of spider venom gland focusing on its cellular structural and functional aspects, and confirms the very specialized nature of the spider venom gland as toxin producer.