Background: Treponema pallidum and HIV are among the most common public health problems in Ethiopia. These infections are interrelated. Treponema pallidum causes genital ulcer which enhances HIV transmission and complicates HIV by causing severe ulcer among HIV-positive individuals. Although Treponema pallidum and HIV have a similar route of transmission, screening services for Treponema pallidum are not available in most of the voluntary counseling and testing centers. Objectives: This study aimed to assess the seroprevalence of Treponema pallidum and sociodemographic factors among HIV-positive and HIV-negative individuals from the voluntary counseling and testing center of St. Paul’s Hospital Millennium Medical College, Addis Ababa, Ethiopia. Methods: In this research, a cross-sectional study was conducted and a total of 292 consecutive samples were collected from the voluntary counseling and testing center of St. Paul’s Hospital Millennium Medical College. Data were collected using an interviewer-administered questionnaire. A blood specimen was tested for the presence of Treponema pallidum using rapid plasma reagin and a rapid test kit for HIV. The data were entered and analyzed using SPSS version 20. Results: The overall prevalence of Treponema pallidum was 5/292 (1.7%). All HIV-positive clients were negative for Treponema pallidum. None of the risk factors were significantly associated with Treponema pallidum. Conclusion: In this study, the prevalence of Treponema pallidum was 5/292 (1.7%). None of the study participants were co-infected with Treponema pallidum and HIV. Recommendation: There was a lack of association between syphilis and HIV in this study, but still the prevalence of Treponema pallidum among voluntary counseling and testing center clients is a public health problem. Therefore, Treponema pallidum infection control strategies should be designed in parallel with HIV control strategy and actions should be taken to avert the problem, including the provision of better health education and screening services at voluntary counseling and testing centers parallel with HIV.