Photoperiod is a pivotal factor in affecting spermatogenesis in seasonal-breeding animals. Transposable elements, a class of DNA sequences that can replicate and translocate in the genome, have regulatory functions during spermatogenesis. However, whether it also functions in photoperiodic spermatogenesis in seasonal breeding animals is unknown. To explore this, we first annotated 5,501,822 transposons in the whole genome of Brandt’s voles (Lasiopodomys brandtii), and revealed that LINEs were the most abundant, comprising 16.61% of the genome. Following closely, SINEs accounted for 10.13%, LTRs for 7.54%, and DNA transposons for 0.70%. Insertion bias analysis revealed that 74.75% of transposons were outside coding genes. Subsequently, we exposed male Brandt’s voles to long-photoperiod (LP, 16 hours/day) and short-photoperiod (SP, 8 hours/day) from their embryonic stages, and obtained testes transcriptome at 4 and 10 weeks after birth. Differential expression and Pearson analysis indicated strongly positive correlations between the expression of differentially expressed retrotransposons and the adjacent genes. KO, KEGG and GSEA results showed that flagellar genes were key target genes regulated by retrotransposons. RT-qPCR results validated the accuracy of the transcriptome using randomly selected six genes (Dnah1, Axdnd1, Ccdc13, Dnah17, Dnah2, Dnali1) and five transposons (LTR/ERVL-MaLR_113132, LINE/L1_1811211, LINE/L1_69082, LINE/L1_662502, SINE/Alu_1213291). RT-PCR results showed that SINE/Alu_1213291 regulates the transcriptional expression of the Dnali1 gene. This study suggests that transposon play a crucial role in the photoperiodic spermatogenesis in Brandt’s voles, especially in the flagella assembly at the late spermiogenesis. Our findings first reveal the regulatory function of transposons in photoperiodic spermatogenesis, providing insights into the role of photoperiod in seasonal reproduction in wild animals.