The protein composition of sputum most faithfully reflects the state of the lungs. The aim of this study was to determine whether relative qualitative and quantitative differences in protein expression of sputum could be related to active pulmonary tuberculosis. Sputum samples were collected from 65 patients with active pulmonary tuberculosis and 38 healthy controls. Comprehensive proteomic approaches were used to profile the proteome changes of host sputum in response to Mycobacterium tuberculosis infection using two-dimensional electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. Mascot software was used to identify proteins from protein databases. Enzyme-linked immunosorbent assay was used to confirm the proteomic results. A total of 62 differentially expressed proteins were identified, among which, 15 proteins were up-regulated and 47 proteins were down-regulated in the tuberculosis sputum compared with the controls. Bacterial protein UqhC was the most increased protein, whereas serum albumin was the most decreased protein in the tuberculosis sputum compared with the controls. The enzyme-linked immunosorbent assay analysis was consistent with proteomic data. Bioinformatics analysis suggested that multiple host cell pathways were involved in the tuberculosis infection processes, including acute phase response, signal transduction, cytoskeleton structure, immune response and so on. In all, for the first time, our results revealed that a number of proteins were differentially expressed during active pulmonary tuberculosis infection. These data will provide valuable clues for further investigation of tuberculosis pathogenesis and biomarkers for detection of active pulmonary tuberculosis infection.