Background <p>Paternal environmental factors can influence offspring development and health through sperm-carried epigenetic information, but the underlying mechanisms are still not fully understood. This study aims to investigate whether paternal vitamin D deficiency can modify the sperm-derived miRNAs and whether it affects the testicular development of the offspring, and to explore the potential molecular mechanisms.</p> Methods <p>Four-week-old male C57BL/6J mice were fed with vitamin D-sufficient or -deficient diet for 16 weeks, then mated with healthy female mice to obtain F1 offspring. High-throughput sequencing of paternal sperm miRNAs and bioinformatics analysis were performed. All offspring were raised to adulthood under standard feeding conditions, and their body weight, reproductive organ development, testicular histomorphology and molecular characteristics were systematically evaluated.</p> Results <p>Paternal vitamin D deficiency induced significant alterations in the sperm miRNA expression profile, with a total of 16 differentially expressed miRNAs being identified. The target genes of these miRNAs were found to be enriched in pathways related to oxidative stress and fibrosis. Compared to the control group, the male offspring of VDD father group exhibited decreased body weight and testicular weight, accompanied by abnormal testicular tissue structure. At the molecular level, the expression of antioxidant-related genes in the offspring testes was down-regulated, while the TGF-β/SMAD2 signaling pathway and fibrosis markers were significantly up-regulated, suggesting enhanced oxidative stress and activation of the fibrotic remodeling.</p> Conclusions <p>This study suggests that paternal vitamin D deficiency may reshape the oxidative stress and fibrosis-related pathways in offspring’s testes accompanied by altering sperm miRNA-mediated epigenetic information.</p>