Background: Provider-initiated testing and counseling (PITC) is defined as “routine HIV testing in a clinical setting as part of a standard program of medical services”. PITC was started in Tanzania in 2007, with the aim of increasing early diagnosis of people with unrecognized HIV infection so that they can access timely prevention and care services in high prevalence settings. However, there is limited information on how PITC is performed in Tanzania especially on health care provider’s knowledge, attitude and practice on PITC since its inception in Tanzania. Objective: To assess the level of knowledge, attitude and practice of health care providers on PITC in the government health facilities of Mbeya City Council. Methodology: A cross sectional study was conducted among 402 consenting health care providers in health care facilities of the Mbeya City Council, between April and May, 2010. A multi-stage sampling was used to select health facilities then health care providers into strata. A self administered questionnaire with both open and closed ended questions was used to collect information. Summarization of data was done using descriptive statistics. A Chi square was used to test the differences between the groups. Ethical approval was obtained from KCM-College ethical committees and informed consent was sought from each study participant. Results: Four hundred and twenty two health care providers were approached and 402 (95%) agreed to participate. The mean age of the participants was 41 years, range (24 - 60 years) and 65% were females. Overall awareness of health care providers on PITC services was high (100%). However, slightly more than one third of health care providers (35%) had inadequate knowledge and attitude towards PITC. Of the 402 participants, 67% (269) reported to have ever offered PITC services, of which 21.9% (59/269) were not actively involved in the provision of PITC. Of the 269 of health care providers who were offering, 29.4% had poor practice, meaning that they did not adhere to the three principles of PITC especially in the area of seeking informed consent. Conclusion: Conclusions emanating from the study findings show that, although PITC is an effective strategy to identify unrecognized HIV infections, missed opportunity to offer the PITC service still occurs at health facilities in Mbeya City Council, as approximately one third of health care providers had never offered the services and others were not actively involved in offering the service. There is a need to improve strategies that, all health care providers receive high quality special training on PITC so that to increase knowledge and equip health facilities with necessary supplies and with adequate human resources. Furthermore, during training health care providers should be emphasized thoroughly on issues regarding informed consent. This can be achieved through development of concrete plans at each health care level with main priority being on capacity building.