Repair and regeneration are key processes for tissue maintenance and their disruption may lead to disease states. Little is known about the molecular mechanisms that underline the repair and regeneration of the digestive tract. The sea cucumber Holothuria glaberrima represents an excellent model to dissect and characterize the molecular events during intestinal regeneration. To study the gene expression profile, cDNA libraries were constructed from normal, 3day, and 7day regenerating intestines of H. glaberrima. Clones were randomly sequenced and queried against the non-redundant (nr) protein database at the National Center for Biotechnology Information. RT-PCR analyses were made of several genes to determine their expression profile during intestinal regeneration. A total of 5173 sequences from three cDNA libraries were obtained. About 46.2%, 35.6%, and 26.2% of the sequences for the normal, 3days, and 7days cDNA libraries, respectively, shared significant similarity with known sequences in the protein database of Genbank, but only present 10% of similarity among them. Analysis of the libraries in terms of functional processes, protein domains, and most common sequences suggest that a differential expression profile is taking place during the regeneration process. Further examination of the expressed sequence tags (ESTs) dataset revealed that 12 putative genes are differentially expressed at significant level (R>6). Experimental validation using RT-PCR analysis reveals that at least three genes (unknown C-4677-1, melanotransferrin, and centaurin) present a differential expression during regeneration. These findings strongly suggest that the gene expression profile varies among regeneration stages and provide evidence for the existence of differential gene expression.