ABSTRACT Background Zika virus (ZIKV) infection during pregnancy can cause severe birth defects in the fetus and is associated with neurodevelopmental abnormalities in childhood. Our objective was to describe ZIKV knowledge and attitudes among pregnant women in Colombia while ZIKV was circulating and whether they predicted the adoption of behaviors to prevent ZIKV mosquito-borne and sexual transmission. Methods We used self-reported data from Zika en Embarazadas y Niños (ZEN), a cohort study of women in early pregnancy across three regions of Colombia during 2017–2018. We used Poisson regression to estimate associations between knowledge, attitudes and previous experience with mosquito-borne infection and preventative behaviors. Results Among 1519 women, knowledge of mosquito-borne transmission was high (1480; 97.8%) and 1275 (85.5%) participants were worried about ZIKV infection during pregnancy. The most common preventive behavior was wearing long pants (1355; 89.4%). Regular mosquito repellent use was uncommon (257; 17.0%). While ZIKV knowledge and attitudes were not associated with the adoption of ZIKV prevention behaviors, previous mosquito-borne infection was associated with increased condom use (prevalence ratio 1.4, 95% CI 1.1 to 1.7). Conclusions Participants were well informed about ZIKV transmission and its health consequences. However, whether this knowledge resulted in behavior change is less certain.