People living with HIV (PWHIV) face negative attitudes that isolate and discourage them from accessing services. Understanding negative attitudes and the social environment can lead to more effective health promotion strategies and programs. However, a scale to measure attitudes has been lacking. We developed and validated attitudes toward PWHIV Scale to examine trends in attitudes toward PWHIV in Quebec in 1996, 2002, and 2010. We also examined the relationship between negative attitudes toward PWHIV, homophobia, and knowledge about HIV transmission. The scale included 16 items and had a five-factor structure: F1 (fear of being infected), F2 (fear of contact with PWHIV), F3 (prejudicial beliefs toward groups at high risk of HIV), F4 (tolerance regarding sexual mores and behaviors), and F5 (social support for PWHIV). The validity and reliability of the scale were assessed and found to be high. Overall, Quebecers had positive attitudes toward PWHIV, with more negative attitudes observed in subgroups defined as male, ≥50 years of age, <14 years of education, higher levels of homophobia, and below-average knowledge about HIV transmission. Scores were stable between 1996 and 2002, and increased in 2010. Negative attitudes were correlated with higher levels of homophobia and lesser knowledge about HIV transmission. The lowest scores for each factor were observed in the same subgroups that had low overall scores on the Attitudes Scale. The findings from this study can be used to intensify interventions that promote compassion for PWHIV, address attitudes toward homosexuality, and encourage greater knowledge about the transmission of HIV in these subgroups.