Macadamia nut(Macadamia integrifolia)is an evergreen nut tree with high economic value. Its kernel is rich in nutrients such as fatty acid and protein, etc. In order to further explore the main regulatory genes related to nutrient formation in M. integrifolia kernels, transcriptomics, gene cloning, fluorescence quantification PCR and bioinformatics techniques were used to screen potential regulatory genes from the kernel transcriptomes of ‘Guire No. 1' and ‘A4', which have significantly different nutrient contents in M. integrifolia kernels. The results were as follows:(1)Transcriptome analysis showed that 1 667 genes were up-regulated and 1 798 genes down-regulated in ‘Guire No. 1' kernel compared with those of ‘A4' kernel; KEGG enrichment analysis showed that the differential genes were mainly in starch and glucose metabolism, amino acid biosynthesis and carbon metabolism.(2)A significant differentially expressed gene gene-LOC122077931 encoding the R2R3-MYB transcription factor MYB44L was discovered. The MiMYB44L gene was cloned in kernels of ‘Guire No. 1' using RACE technology, which was 1 165 bp in length, 999 bp in ORF in length, and encoded 332 amino acids.(3)Bioinformatics analysis confirmed the presence of the SANT domain in the MiMYB44L protein, a hallmark feature of the R2R3-MYB family. The protein lacked both a signal peptide and a transmembrane domain but featured phosphorylation sites.(4)The protein contents in kernels of 10 M. integrifolia varieties were determined. And it was found that the expression of MiMYB44L gene in M. integrifolia varieties with high protein content was significantly higher than that in varieties with low protein content, and the overall correlation coefficient was 0.54, reaching a extremely significant level. The results of this study provide theoretical guidance for in-depth analysis of the regulatory mechanism of MiMYB44L gene in the formation of protein content in M. integrifolia.