Diaphania glauculalis (Guenée) is a defoliating pest of the Laran tree, Anthocephalus chinensis (Lamk.) Rich. ex Walp., which is an important tree species for reforestation efforts in tropical and subtropical regions. Currently, little information is available about the calling behavior and pheromone production of D. glauculalis females, but such information is important in developing pheromone-based monitoring systems for the pest. Thus, female calling behavior and its correlation with pheromone production was studied using electroantennogram (EAG) technology, qualitative analysis of gas chromatography-mass spectrometry (GC-MS), and Y-tube olfactometer assays. Calling females release a sex pheromone from a gland located on the dorsal membrane between the eighth and ninth abdominal segments, as observed with a stereomicroscope. Peak calling behavior occurred with females 3 d after emergence and 6 h into the scotophase. Pheromone release typically increased with the female age and scotophase and then decreased gradually. GC-MS analysis showed that the amount of pheromone in extracts from the pheromone glands were greatest at the onset of the calling behavior and decreased thereafter. Y-tube olfactometer assays confirmed that pheromone gland extracts at 6 h into the scotophase elicited stronger attractiveness in comparison to 5, 7, and 8 h after emergence.