The institution of schooling is a key socializing agent, and teachers' attitudes toward gender and sexual diversity play a crucial role in the education of all children, shaping in particular the school experience of LGBT + children and youth. Our research used a survey of Early Childhood and Primary teachers (N = 182) and university students studying these degree programs (N = 1,133) in northwestern Spain (Galicia) to analyze transphobic and homophobic attitudes, and their relationship with other professional and academic variables, such as years of experience or study, specialization or degree program, and place of work or study. Results showed that practicing teachers generally displayed less tolerant attitudes than students studying to be teachers, although tolerance increased with the numbers of years of teaching experience. We also found differences among students according to their course of study, with those preparing to teach at the Early Childhood Education level showing more positive attitudes, and also depending on the location of their university campus, suggesting that the program design and local sociocultural context may be contributing factors.