This study assessed the protective effect of the salivary pellicle formed in vivo during 24 h or 7 days against demineralization of bovine enamel caused by citric acid. In addition, the influence of acid treatment on the behavior of the pellicle was investigated. Enamel specimens with and without in vivo pellicles were immersed in citric acid (0.1, 1.0%) over 30, 60, and 300 s, and processed for scanning (SEM) and transmission electron microscopy (TEM), as well as for measurement of surface microhardness (SMH). Specimens coated with the in vivo formed pellicles revealed less extensive erosive demineralization of the enamel surface compared to uncovered enamel specimens. SEM analysis and SMH results did not indicate distinct differences between erosive surface alterations on enamel slabs covered with 24-hour pellicles and on those covered with 7-day pellicles. TEM analysis showed that the pellicle layer was dissolved in part from the enamel surface due to acid exposure. However, pellicle residues could be detected by TEM in all specimens, even after 5-min exposure to 1.0% citric acid. It is concluded that the in vivo salivary pellicle can resist the acidic action to some extent and provides protection to the underlying enamel surface against erosive destruction caused by short-term action of citric acid.