<p>The machining errors are affected by positioning errors. However, the relationship between positioning error and machining error has not been fully comprehended due to the complexity of the positioning error distribution within the machining range of machine tool. The purpose of this paper is to elucidate the effects of positioning error of different distributions on machining error, and thereby improve the machining accuracy of machine tools. First, a test piece with multiple features is designed, positioning error measurement and test piece machining are conducted on four machine tools with different structures. Then, uncertainty assessment is introduced to examine the factors that affect measurement accuracy in the positioning error measurement process of laser interferometers, ensuring the validity of measurement results and the rationality of subsequent data analysis. Finally, on the basis of meeting the uncertainty assessment requirements in the measurement results, the effects of positioning error on machining error are investigated combining with actual machining, and the manifestation of the tool tip motion trajectory deviation caused by positioning errors on machining errors is clarified. The uncertainty assessment results suggest that incorrect material thermal compensation has the most significant effect on the measurement results, and actual machining results reveal that the distribution of positioning error, as well as the relative position between the test piece and tool coordinate system, will affect the machining error. This paper provides a practical reference for engineers to predict machining errors based on positioning errors.</p>