Low-income countries with high Tuberculosis burden have few reference laboratories able to perform TB culture. In 2006, the Zanzibar National TB Control Programme planned to decentralize TB diagnostics. The Italian Cooperation Agency with the scientific support of the "L. Spallanzani" National Institute for Infectious Diseases sustained the project through the implementation of a TB reference laboratory in a low-income country with a high prevalence of TB. The implementation steps were: 1) TB laboratory design according to the WHO standards; 2) laboratory equipment and reagent supplies for microscopy, cultures, and identification; 3) on-the-job training of the local staff; 4) web- and telemedicine-based supervision. From April 2007 to December 2010, 921 sputum samples were received from 40 peripheral laboratories: 120 TB cases were diagnosed. Of all the smear-positive cases, 74.2% were culture-positive. During the year 2010, the smear positive to culture positive rate increased up to 100%. In March 20, 2010 the Ministry of Health and Social Welfare of Zanzibar officially recognized the Public Health Laboratory- Ivo de Carneri as the National TB Reference Laboratory for the Zanzibar Archipelago. An advanced TB laboratory can represent a low cost solution to strengthen the TB diagnosis, to provide capacity building and mid-term sustainability.