Chlamydia trachomatis is an obligate intracellular bacterium causing infections of the eyes, urogenital and respiratory tracts. Asymptomatic, repeat and chronic infections with C. trachomatis are common in the urogenital tract potentially causing severe reproductive pathology. Animal models of infection and epidemiological studies suggested the gastrointestinal tract as a reservoir of chlamydiae and as a source of repeat urogenital infections. Thus, we investigated the growth characteristics of C. trachomatis in human intestinal epithelial Caco-2 cells and the infection-induced defensin production. Immunofluorescence staining and transmission electron microscopy showed the presence of chlamydial inclusions in the cells. Chlamydial DNA and viable C. trachomatis were recovered from Caco-2 cells in similar quantity compared to that detected in the usual in vitro host cell of this bacterium. The kinetics of expression of selected C. trachomatis genes in Caco-2 cells indicated prolonged replication with persisting high expression level of late genes and of heat shock protein gene groEL. Replication of C. trachomatis induced moderate level of β-defensin-2 production by Caco-2 cells, which might contribute to avoidance of immune recognition in the intestine. According to our results, Caco-2 cells support C. trachomatis replication, suggesting that the gastrointestinal tract is a site of residence for these bacteria.