Epididymitis is a highly prevalent disease in the outpatient urologic clinic and a relevant factor in male infertility. Bacterial infections, including sexually transmitted bacteria and common uropathogens, represent the most common etiological factors of epididymitis. Nevertheless, the pathophysiological mechanisms underlying the onset, natural history, and outcomes of bacterial epididymitis remain poorly understood. In this regard, rodent models are valuable tools for investigating the mechanistic responses of the epididymis when challenged with various inflammatory or infectious stimuli. Studies in rats and mice showed that bacterial-derived pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from Gram-negative and Gram-positive bacteria, respectively, trigger acute inflammation in the epididymis. The severity of these inflammatory responses differs according to the epididymal region affected and is associated with poor sperm parameters. Herein, two distinct surgical approaches are described in detail for inducing epididymitis in mice through the injection of PAMPs in the interstitial compartment of the initial segment or the luminal compartment of the vas deferens toward the cauda epididymidis, thus allowing investigation of the inflammatory responses of the epididymal proximal and distal regions. It is expected that these experimental models of epididymitis will enable future studies aimed at advancing the understanding of the mechanisms governing the epididymal responses to invading pathogens, opening novel ways for therapeutic interventions to mitigate the repercussions of epididymitis on male fertility.