Objective: To determine the perceived difficulties in providing HIV/AIDS services among public health nurses and to identify their correlates, we carried out a cross-sectional study in the Kinki region of western Japan. Methods: Structured self-administered questionnaires were distributed to all public health nurses in the region, and 1535 valid questionnaires were retrieved (valid response rate 78.7%). Results: More than half of the participants (52.8%) reported difficulties with HIV/AIDS services. The factors associated with perceived difficulties were having a negative attitude towards consultations on sexual matters (adjusted odds ratio [AOR] 2.2, p < 0.001), a perceived lack of encounters with homosexual people and people with HIV/AIDS during practice (AOR 1.6, p = 0.002 and AOR 1.8, p < 0.001), poor knowledge of sexual diversity (AOR 2.0, p < 0.001), lack of training in sexual diversity in public health nursing education (AOR 1.4, p = 0.016), and low permissiveness of the diversity of sexual behavior (AOR 2.0, p < 0.001). Conclusions: Overall, our results suggest that nursing and public health nursing education in Japan should cover sexual issues and HIV/AIDS in a more systematic way.