Doxycycline postexposure prophylaxis (doxy-PEP) could significantly reduce syphilis incidence. However, the increase in intermittent doxycycline usage might select resistant Treponema pallidum strains. To assess whether resistance to doxycycline could be induced in this pathogen, we exposed the SS14 strain in vitro, both intermittently and continuously, to a subbactericidal doxycycline concentration that still exerts antibiotic pressure. During and after each exposure experiment, we assessed the doxycycline minimal inhibitory concentration in test and control treponemes and performed whole-genome sequencing, concluding that no resistance developed. This work suggests that doxycycline-resistant T. pallidum is not an immediate threat for doxy-PEP implementation.