Background Macadamia species are valuable nut trees, and physiological fruit abscission severely reduces their yield. An inadequate carbohydrate supply is the main cause of physiological abscission in macadamia; however, the molecular mechanism of their fruit abscission has not been fully characterized.Therefore, this study aimed to determine the molecular mechanisms involved in physiological fruit abscission in macadamia via comparative transcriptome analysis. Results Macadamia fruits were exposed to carbohydrate starvation stress by girdling plus defoliation (GPD) treatment, which induced mass fruit abscission within a few days. Changes in the soluble sugar content of the fruit and the transcript levels of genes in the abscission zone were analyzed and identified during the abscission process. Compared with that in the control group, the increase in the fruit abscission rate after GPD treatment was related to a decrease in the soluble sugar content, especially the sucrose and glucose contents, in the pedicel. At 1, 2, 3, and 5 days after GPD treatment, 2093, 2267, 5099, and 1235 differentially expressed genes (DEGs), respectively, were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the functions and pathways of these DEGs were related to hydrolases and transferases, carbohydrate metabolism, plant hormone synthesis, and signal transduction. Many DEGs involved in amino acid metabolism, phenylpropanoid and flavonoid biosynthesis directly responded to carbohydrate starvation signals. Several transcription factor families, including bHLH, ERF, MYB, NAC, and WRKY, were found to play key roles in gene transcriptional regulation during macadamia fruit abscission. Weighted gene co-expression network analysis was used to analyze the RNA sequencing data to predict relationships between DEGs.Ten modules were identified, among which the pink, blue, and red modules were significantly correlated with samples after GPD treatment for 1, 3, and 5 days, respectively, and contained 74, 1518, and 111 DEGs, respectively. Additionally, the expression patterns of 12 DEGs were analyzed via quantitative real-time PCR to verify the accuracy of the RNA sequencing data. Conclusions This study identified candidate genes for further research aimed at reducing fruit abscission to increase yield in macadamia.The findings also provide molecular biology data that contribute to a deeper understanding of the regulatory mechanism governing physiological fruit abscission in macadamia.