The objective of this study was to identify determinants of syphilis infection among pregnant women attending antenatal care in hospitals in the Wolaita zone, Southern Ethiopia,2020. An unmatched facility-based case-control study was conducted among pregnant women who received antenatal care at four randomly selected hospitals from September 1 to October 30, 2020. A two-stage sampling technique was used in the selection of hospitals and study participants. The data were collected from the participants using a pre-tested structured questionnaire and analyzed using STATA Release 15. Bivariate and multivariate logistic regression analyses were used to determine syphilis infection determinants. Crude and adjusted odds ratios were used for each explanatory variable with a 95% confidence level. A statistically significant association was declared when a p-value was less than 0.05. A total of 296 (74 cases and 222 controls) pregnant women participated, with a recruitment rate of 97.4%. In multivariate logistic regression, the likelihood of developing a maternal syphilis infection was higher in pregnant women who had more than one-lifetime sexual partner [AOR = 3.59, 95% CI (1.09-11.71)]; a history of sexually transmitted infections [AOR = 3.46, 95%CI (1.32-9.08)] and used a substance [AOR = 3.39, 95%CI (1.31-8.77)]. Sexual-related factors continued to be a major determinant of syphilis in pregnant women. The results suggest that there is a need to promote safe sexual behavior, raise awareness about the risk of STIs, and early diagnosis and treatment of STIs to control syphilis infection, and necessary to make the antenatal care service comprehensive for pregnant women.