Chlamydia persistence is a viable but non-replicative stage, induced by several sub-lethal stressor agents, including beta-lactam antibiotics. So far, no data about the connection between doxycycline and chlamydial persistence has been described in literature. We investigated the ability of doxycycline to induce C. trachomatis (CT) persistence in an in vitro model of epithelial cell infection (HeLa cells), comparing the results with the well-established model of penicillin-induced persistence. The effect of doxycycline was explored on 10 different CT strains by analysing (i) the presence of aberrant inclusions, (ii) chlamydial recovery, (iii) the expression of different chlamydial genes (omcB, euo, Ct110, Ct604, Ct755, HtrA) and (iv) the effects on epithelial cell viability. For each strain, the presence of foreign genomic islands responsible of tetracycline resistance was excluded. We found that low doses of doxycycline can induce a condition of CT persistence. For concentrations of doxycycline equal to 0.03-0.015 mg/L, CT inclusions are smaller and aberrant and CT cycle is characterized by the presence of viable but non-dividing RBs with the complete abolishment of chlamydial cytotoxic effect. Infectious EBs can be recovered after removal of the drug. During doxycycline-induced persistence, the expression of the late gene omcB is decreased, indicating the blocking of RB-to-EB conversion. Conversely, as for penicillin G, a significant up-regulation of the stress response HtrA gene is found in doxycycline-treated cells. This study provides a novel in vitro cell model to examine the characteristics of doxycycline-induced persistent CT infection.