Provider-initiated testing and counseling (PITC) is a routine HIV counseling and testing offered to adult clients attending at health care facilities. In Tanzania PITC started in 2007, with the aim of increasing early HIV diagnosis and timely access to care, treatment and support services. The objective of this study was to assess the level of knowledge and practice of health care providers on PITC. This cross sectional study was conducted in April-May 2010 among nurses and clinicians working in the government health care facilities in Mbeya City, south-western Tanzania . Self-administered questionnaire was used to obtain relevant information from the study participants. A total of 402 (95% response rate) care providers were interviewed. Their mean (± SD) age was 41 ± 9.5 years. The majority 304 (76%) were nurses. Their duration of practice ranged from 4 months to 39 years. All the care providers reported to be aware of PITC services. However, 35% of them had inadequate knowledge of PITC and 269 (67%) reported to have ever offered PITC services. Median number of clients attended per week was 3 (IQR, 1-6 clients). Participants who had attended special training on PITC were 6-fold more likely to offer PITC services [OR = 6.2, 95% CI = 3.7-10.2; P <0.001] than those who had not attended. In conclusion more than one-third of health providers do not routinely offer PITC in Mbeya City, leading to missed opportunity for early HIV diagnosis. On job and in-service training to improve PITC knowledge and supportive supervision for those trained is required.