The professional attitude of health care workers (HCWs) who serve HIV/AIDS patients and drug users is important in implementation of the harm reduction program (HRP). This study was to explore the causal relationships between education and training, AIDS-related knowledge, attitude of supporting methadone maintenance treatment (MMT), risk perception, and professional attitude of HCWs toward serving HIV/AIDS patients and drug users. We distributed a self-administered questionnaire to HCWs who have served HIV/AIDS patients and drug users due to work in Taoyuan, northern Taiwan. Structural equation modeling (SEM) was used to test various pathways regarding the professional attitudes of HIV/AIDS patients and drug users among HCWs. A total of 218 HCWs were eligible for this study. The dual pathway model was emerged: (1) have attended education and training courses regarding to HRP positively and significantly affects professional attitude via the attitude of supporting MMT. The correlation (r) was 0.27 between education and training and the attitude of SMMT, and was 0.42 between the attitude of SMMT and professional attitude. (2) AIDS-related knowledge negatively and significantly affects professional attitude via risk perception of contracting HIV. The correlation was -0.22 between AIDS-related knowledge and risk perception, and was -0.25 between risk perception and professional attitude. Various fit indices confirmed a reasonable and acceptable fit of the model. Balance theory and approach-avoidance conflict may partially explain the dual pathways of professional attitude of HCWs toward serving HIV/AIDS patients and drug users. Our result suggests that, among HCWs, education and training courses regarding to HRP are important in increasing the attitude SMMT and AIDS-related knowledge directly, thus, professional attitude serving HIV/AIDS patients and drug users can be enhanced indirectly.