Macrophages are the major type of immune cell in the testis of both humans and rodents. Testicular macrophages (TMs) play critical roles in maintaining the testicular microenvironment, such as Leydig cell-dependent hormone production, spermatogenesis, and immune balance. A substantial number of studies have used rodent models to investigate the functions of TMs with various methods and harvest macrophages from the testis. Studies have demonstrated that enzyme digestion, an essential part of these methods, can improve the number and purity of TMs while unavoidably altering the immunoprofile of macrophages, which is detrimental for further study in terms of immune investigation. Here, we modified the existing method of microglia isolation and set up a novel method without the enzyme digestion step to isolate TMs. According to the characteristics of testicular tissue looseness and the physical and biological characteristics of macrophages, by combining mechanical separation, gradient centrifugation, and immuno-magnetic bead selection, we can effectively avoid the enzymatic digestion of testis tissue and maintain the immune characteristics of macrophages. Additionally, we verified the purity of TM with flow cytometry (FC) at approximately 91-95%, and the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) was lower than that isolated with enzyme digestion. In contrast to the traditional method, this novel protocol can assist those who have no convenient access to fluorescence-activated cell sorting (FACS) to isolate a sufficient number of TMs and, most importantly, avoid altering the immunoprofile of TMs without enzyme digestion.Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.