ABSTRACT: Background: Acute sprint exercise is a time-efficient physical activity that improves cardiorespiratory fitness in younger and middle-aged adults. Growing evidence has demonstrated that acute sprint exercise provides equal to or superior health benefits compared with moderate-intensity continuous training, which will dramatically increase aerobic capacity, insulin sensitivity, and muscle capillarization. Although the beneficial effects of acute sprint exercise are well documented, the mechanisms behind how acute sprint exercise prevents disease and benefits health are less understood. Method: We obtained differentially expressed genes in muscle (vastus lateralis) from men and women before and after an acute sprint exercise. Then, we identified hub genes from the protein–protein interaction (PPI) network of differentially expressed genes (DEGs) and key transcription factors in men and women related to acute sprint exercise. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses are performed on DEGs and sex-biased genes, respectively. Results: First, we identified 127 sexually dimorphic genes in men (90 upregulated and 37 downregulated) and 75 genes in women (90 upregulated and 37 downregulated) in response to acute sprint exercise. Second, CEBPB, SMAD3, and CDKN1A are identified as the top three hub genes related to men-biased genes. Accordingly, the top three hub genes related to women-biased genes are JUN, ACTB, and SMAD7. In addition, CLOCK, ZNF217, and KDM2B are the top three enriched transcriptional factors in men-biased genes, while XLR, SOX2, JUND, and KLF4 are transcription factors enriched most in women-biased genes. Furthermore, based on GO and KEGG enrichment analyses, we identified potential key pathways in regulating the exercise-related response in men and women, respectively. Conclusion: In this study, we found the difference in gene expression and enrichment pathways in muscle in men and women in response to acute sprint exercise. These results will shed new light on the mechanism underlying sex-based differences in skeletal muscle remodeling and metabolism related to acute sprint exercise, which may illustrate the mechanisms behind how acute sprint exercise prevents disease and benefits health.