Streptococcus pneumoniae is a Gram-positive bacterial pathogen causing a variety of diseases, including otitis media, bacteraemia and meningitis. Although copper is an essential trace metal for bacterial growth, high intracellular levels of free-copper are toxic. Copper resistance has emerged as an important virulence determinant of microbial pathogens. In this study, we determined the minimum inhibition concentration of copper for the growth inhibition of S. pneumoniae. Two-dimensional-electrophoresis coupled with mass spectrometry was applied to identify proteins involved in copper resistance of S. pneumoniae. In total, forty-four proteins with more than 1.5-fold alteration in expression (p < 0.05) were identified. Quantitative reverse transcription PCR was used to confirm the proteomic results. Bioinformatics analysis showed that the differentially expressed proteins were mainly involved in the cell wall biosynthesis, protein biosynthesis, purine biosynthesis, pyrimidine biosynthesis, primary metabolic process, and the nitrogen compound metabolic process. Many up-regulated proteins in response to the copper treatment directly or indirectly participated in the cell wall biosynthesis, indicating that the cell wall is a critical determinant in copper resistance of S. pneumoniae.