Cholesterol is an important part of the human diet. The relationship and molecular mechanisms between intracellular cholesterol and male infertility are unclear. The purpose of this study was to evaluate the role of low-density lipoprotein receptor (LDLR) in male infertility. Both wild-type (WT) and LDLR heterozygous deletion (LDLR+/-) male Golden Syrian hamsters were fed either a high-fat diet (HFD) or a normal diet (ND). Plasma biochemistry, serum hormone, testicular histopathology, mRNA and protein expression of AMPK/Sirt1/PGC-1α in both testicular tissue and isolated Leydig cells (LCs) were measured. Compared with the ND animals, the WT HFD hamsters developed dyslipidemia at three weeks with lipid droplets deposited in LCs, testosterone decreased at four weeks (0.440 ± 0.264 ng/ml vs. 2.367 ± 1.236 ng/ml), the number of the Sertoli cells decreased (21.578 ± 2.934/one tubule vs. 25.733 ± 3.424/one tubule), the seminiferous epithelium became thinner (0.0813 ± 0.01729 mm vs. 0.0944 ± 0.0138 mm), testicular atrophy and AMPK/Sirt1/PGC-1α pathway downregulated at five weeks. All these changes persisted until the end of the study. LDLR+/- alleviated all of the above changes by downregulating the cellular influx of cholesterol induced by HFD except for higher hyperlipidemia. In summary, excessive intracellular cholesterol inactivates AMPK/Sirt1/PGC-1α pathway firstly in LCs and then in both Sertoli and spermatids. Cholesterol toxicity was LDLR dependent.Copyright © 2023 Elsevier B.V. All rights reserved.