For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and harvest of the organisms from rabbit testes. In 2018, we described a method to continuously cultivate T. pallidumin vitro. This system utilizes co-culture with rabbit epithelial cells (Sf1Ep cells) in a serum-containing tissue culture medium called TpCM-2. The T. pallidum doubling time in culture is similar to that estimated to occur during natural infection (about 33-45 h). The organism can be cultured continuously with a standard passage time of 1 week in a low oxygen (1.5%) environment at 34 °C. This article contains the protocols for culturing T. pallidum, methods for growing and maintaining the required tissue culture cells, and the technique for generating isogenic strains by limiting dilution. The ability to grow T. pallidum in vitro provides new experimental avenues to study and understand this enigmatic organism.