The presence of translucent eggshells is a type of egg quality issue that impacts egg sales. While many researchers have studied them, the exact mechanisms behind their formation remain unclear. In this study, we conducted a transcriptomic differential expression analysis of the isthmus region of the oviduct in both normal egg- and translucent egg-laying hens. The analysis revealed that differentially expressed gene pathways were predominantly concentrated in the synthesis, modification, and transport of eggshell membrane proteins, particularly collagen proteins, which provide structural support. These findings suggest that variations in the physical structure of the eggshell membrane, resulting from changes in its chemical composition, are the fundamental cause of translucent eggshell formation. This research provides a theoretical reference for reducing the occurrence of translucent eggs.