Dietary restriction and/or exercise has been shown to have multiple benefits for health. However, its effects on reproductive health and the mechanisms by which it regulates reproductive function remain unclear. Here, to evaluate its effects on spermatogenesis and sperm function, rats were divided into 4 groups: ad libitum-fed sedentary control, dietary restriction (DR), exercise training (ET), and dietary restriction plus exercise training (DR+ET) groups. Results indicated that body weight, epididymal fat pad weight, and sperm counts were significantly reduced in the DR, ET, and DR+ET groups. Moreover, sperm motility and capacitation-associated protein tyrosine phosphorylation were suppressed in the DR and DR+ET groups, but not the ET group. Microarray analysis revealed that the number of downregulated genes was higher than that of upregulated genes in the DR and/or ET groups. About half of the downregulated genes are common after exercise training and/or diet restriction. Gene ontology analysis showed that downregulated genes in the DR, ET, and DR+ET groups affected spermatogenesis through overlapping pathways, including glucocorticoid, corticosteroid, extracellular structure organization, and estradiol responses. Our findings suggest that diet restriction and/or exercise training may present potential risks to male reproductive dysfunction by disrupting normal gene expression patterns in the testis. Novelty: Dietary restriction and/or exercise can lead to the damage of spermatogenesis as well as sperm maturation. Sperm functional changes are more sensitive to dietary restriction than exercise training. Dietary restriction and exercise impair spermatogenesis through overlapping biological pathways in the testis.