Increasing number of people suffering from HIV/AIDS influenced healthcare sectors. Nurses play a critical role in caring the HIV positive patients. People living with HIV/AIDS usually experienced excessive negative attitudes from health care workers in Iran. The aim of this study was to assess the knowledge and attitude of nurses related to care of patients with HIV/AIDS. This was a cross-sectional survey of 331 nurses from Golestan Hospital in Ahvaz, Iran. Self-administered questionnaire was used to determine the knowledge and attitude of nurses. The study showed a moderate level of knowledge among nurses (55.6% have good knowledge) which is significantly associated with nurses education background, nurses departments and having experience of caring for HIV/AIDS patients. Fear of contagion was great among nurses (76%). Slightly more than half of the nurses had unfavorable attitude towards caring for HIV/AIDS patients (53%). There were several areas of deficiency in knowledge and attitude towards caring for HIV/AIDS patients among nurses. Implementing specific and focused educational programme on HIV/AIDS for nurses is necessary in the health care system in Iran.