Despite recent advances in automated laboratory systems, the Gram stain is still one of the most important tools in clinical microbiology, especially for laboratories with limited resources. With the development of molecular techniques and rapid immunological methods, it now seems that less attention has been paid to the Gram stain compared with before. This review describes the role that the Gram stain plays in the rapid presumptive diagnosis, quality evaluation of clinical samples, and antimicrobial management. Some discussion is also presented in relation to the interpretation of Gram stain and the quality control of Gram staining method.