Bacterial infections, particularly uropathogenic E . coli (UPEC), contribute substantially to male infertility through tissue damage and subsequent fibrosis in the testis and epididymis. The role of testicular macrophages (TMs), a diverse cell population integral to tissue maintenance and immune balance, in fibrosis is not fully understood. Here, we used single-cell RNA sequencing in a murine model of epididymo-orchitis to analyze TM dynamics during UPEC infection. Our study identified a marked increase in S100a4 ⁺ macrophages, originating from monocytes, strongly associated with fibrotic changes. This association was validated in human testicular and epididymal samples. We further demonstrated that S100a4 ⁺ macrophages transition to a myofibroblast-like phenotype, producing extracellular matrix proteins such as collagen I and fibronectin. S100a4, both extracellular and intracellular, activated collagen synthesis through the TGF-β/STAT3 signaling pathway, highlighting this pathway as a therapeutic target. Inhibition of S100a4 with niclosamide or macrophage-specific S100a4 KO markedly reduced immune infiltration, tissue damage, and fibrosis in infected murine models. Our findings establish the critical role of S100a4 ⁺ macrophages in fibrosis during UPEC-induced epididymo-orchitis and propose them as potential targets for antifibrotic therapy development.