The vibration analysis method has been widely used to detect faults in the components of a machine. For a linear positioning stage, its vibration characteristics are closely related to and affected by the preload of linear guides. To clarify the meaning of the vibration signals coming out from an abnormal machine, in this study we aimed to investigate the influence of a faulty linear guide on the vibration and acoustic characteristics of a positioning stage through experimental and finite element modeling approaches. Experimental measurements indicate that stages equipped with abnormal or damaged linear guides indeed exhibit different vibration behavior compared to stages with intact guides. According to finite element analysis, the variation in vibration characteristics can be attributed to the changes in the structural stiffness of linear guides because of rolling ball defects. This phenomenon was also found in the acoustic characteristics measured for stages under various preloaded conditions, providing further correlations between the variations in acoustic characteristics and changes in structural stiffness. The present results clearly show that faulty conditions of linear guides can be effectively detected by means of monitoring the vibration and acoustic characteristics of the stage.