Organotypic models to investigate host-microbiome interactions are still a challenge for the field of tissue engineering. This is particularly the case for organs such as the urethra. Several cell line, animal, and tissue models are available to study Chlamydia trachomatis infections, but none fully reflects natural infection in native human tissue. Therefore, we developed an organotypic reconstructed human urethral model (RhU) to study invasive and noninvasive strains of C. trachomatis. Primary urethra cells were used to reconstruct epithelium on a fibroblast populated collagen-fibrin hydrogel, yielding a RhU. Immunohistochemistry was used to compare RhU with native urethral tissue and to visualize the location of C. trachomatis bacteria in RhU after 10-day exposure. RhU closely resembled native urethral tissue with respect to proliferation and differentiation markers (keratins 6, 10, 13, 17, involucrin, SKALP [skin-derived antileucoproteinase], vimentin, and CD31). Exposure of RhU to noninvasive and invasive C. trachomatis strains revealed relevant differences in infection ability because inclusions were observed (indicating active infection) in the epithelial layer after 10 days exposure only to the invasive strain. The noninvasive strain remained localized on the surface of the epithelial layer. Human primary urethral fibroblasts and keratinocytes can be used to construct RhU that closely resembles native tissue and can be used to investigate active C. trachomatis infections. RhU provides a promising model to investigate host-microbiome interactions such as, but not limited to, the human pathogenesis of C. trachomatis.