BACKGROUND: Pre-exposure prophylaxis (PrEP) is an effective human immunodeficiency virus (HIV) prevention intervention, but its access and use are suboptimal, especially for women. Healthcare providers provision of PrEP is a key component of the initiative. Although training gaps are an identified barrier, evidence is lacking regarding how to tailor trainings for successful implementation. Title X family planning clinics deliver safety net care for women and are potential PrEP delivery sites. To inform provider training, we assessed PrEP knowledge, attitudes, and self-efficacy in the steps of PrEP care among Title X providers in the Southern United States. METHODS: We used data from providers in clinics that did not currently provide PrEP from a web-based survey administered to Title X clinic staff in 18 Southern states from February to June 2018. We developed generalized linear mixed models to evaluate associations between provider-, clinic-, and county-level variables with provider knowledge, attitudes, and self-efficacy in PrEP care, guided by the Consolidated Framework for Implementation Research. RESULTS: Among 351 providers from 193 clinics, 194 (55%) were nonprescribing and 157 (45%) were prescribing providers. Provider ability to prescribe medications was significantly associated PrEP knowledge, attitudes, and self-efficacy. Self-efficacy was lowest in the PrEP initiation step of PrEP care and was positively associated with PrEP attitudes, PrEP knowledge, and contraception self-efficacy. CONCLUSIONS: Our findings suggest that PrEP training gaps for family planning providers may be bridged by addressing unfavorable PrEP attitudes, integrating PrEP and contraception training, tailoring training by prescribing ability, and focusing on the initiation steps of PrEP care.