We assessed the impact of an HIV care mentorship intervention on knowledge, attitudes, and practices with nurses and people living with HIV (PLWH) in Canada. We implemented the intervention in two urban and two rural sites with 16 mentors (eight experienced HIV nurses and eight PLWH) and 40 mentees (nurses with limited HIV experience). The 6- to 12-month intervention included face-to-face workshops and monthly meetings. Using a mixed-methods approach, participants completed pre- and postintervention questionnaires and engaged in semistructured interviews at intervention initiation, mid-point, and completion. Data from 28 mentees (70%) and 14 mentors (87%) were included in the quantitative analysis. We analyzed questionnaire data using McNemar test, and interview data using content analysis. Results indicated positive changes in knowledge, attitudes, and practices among nurse mentees, with qualitative interviews highlighting mechanisms by which change occurred. Mentorship interventions have the potential to engage and educate nurses in HIV treatment and care.