The effects of probiotics supplementation on the reproductive function have been evaluated in many species, but no study has evaluated the changes in the gut microbiome along with the sperm quality changes simultaneously. This study evaluated the effects of dietary supplementation with probiotics on the gut microbiome, sperm quality and gene expression, along with possible correlations between these parameters in dogs. The dogs were supplemented with Lactobacillus rhamnosus for six weeks, and fecal and semen samples were collected at 0, 3, and 6 weeks. Fecal samples were assessed using 16S Metagenomic Sequencing for gut microbiome analysis; and semen samples were analyzed using computer-assisted sperm analysis, DNA and acrosome integrity assessment, viability and morphology assessment, and real-time PCR. The analyses suggested that probiotic supplementation improved kinematic parameters, viability, DNA and acrosome integrity, and morphology of sperms. The mRNA levels of genes associated with fertility, DNA repair and integrity, and antioxidation were also upregulated. The sperm parameters were positively correlated with the relative abundance of Actinobacteria, Allobaculum, Phascolarctobacterium and Catenibacterium, and negatively correlated with Faecalibacterium and Streptococcus. Taken together, the sperm quality enhancement through the gut-testis axis may be due to a change in the gut microorganisms populations. © 2023. The Author(s), under exclusive licence to Springer Nature B.V.