Introduction- Toxoplasma has been implicated as one of the most common opportunistic infection and has posed many diagnostic and therapeutic challenges for clinicians treating HIV/AIDS patients. Seroprevalence of toxoplasmosis in India in general population ranges from 16.3% to 30.8%. , toxoplasmosis has been implicated as one of the most important opportunistic infections in AIDS patients of which toxoplasmic encephalitis is the most common clinical disease, most frequently causing focal intracerebral lesions in these patients. So the present study was conducted to assess seropositivity status of toxoplasmosis in HIV/AIDS patients attending the antiretroviral centre (ART) in a tertiary care hospital in Nagpur city (Maharashtra). Material and Methods- The present Prospective Hospital Based Study was conducted in the Department of Microbiology from Nov 2010 to Oct. 2012 in ART clinic, Medicine-wards of Tertiary Care Hospital of Nagpur city, (Maharashtra) India. 362 HIV positive patients filtered through following criteria, either hospitalized or coming to ART clinic were included in the study. After taking written informed consent, detailed clinical history and relevant investigations of each patient was done and findings were noted as per standard predesigned and pretested the clinical proforma. Results- Out of these 362 HIV positive patients, 226 were males (62.43%) and 136 were females (37.57%). Heterosexual route was found to be the most common mode of transmission in 332(91.71%) patients followed by blood transfusion in 3(0.83 %) cases. Antitoxoplasma antibody (IgM and IgG) was found to be significantly higher in test group than control group. (p<0.0001) Conclusion- The prevalence of toxoplasmosis in HIV positive patients was 23.48%. Therefore these high risk patients should be screened regularly for HIV & toxoplasmosis.