Purpose <p>Sperm mature as they transit through the epididymis, during which they gain progressive motility and the capability to fertilize oocytes. Autophagy plays a significant role in the regulation of testicular spermiogenesis. Autophagy-related gene 5 (<i>Atg</i>5<i>)</i> is recognized as a crucial regulator of autophagy progression. However, the role of <i>Atg</i>5 in regulating epididymal sperm maturation has yet to be elucidated.</p> Methods <p>To investigate the function of <i>Atg</i>5 in regulating epididymal sperm maturation, <i>Atg</i>5 conditional knockout mice were generated using Cre/LoxP technology, and sperm quality and fertility were assessed. Comprehensive quantitative analyses and bioinformatics evaluations of the proteome in segments 4–5 of the caput epididymis and sperm from the cauda epididymis were conducted.</p> Results <p><i>Atg</i>5 deletion reduced autophagy in the caput epididymis but did not significantly affect sperm motility. Quantitative proteomics via data-independent acquisition (DIA) and subsequent bioinformatics analysis revealed significant alterations in the expression of several sperm proteins. Among these, the upregulated proteins were strongly associated with <i>Atg</i>5-independent surrogate autophagy, while the downregulated proteins were linked to sperm maturation. Additionally, qRT-PCR assays demonstrated that seven genes closely related to autophagy and sperm quality were differentially expressed in the 4–5 segments of the caput epididymis.</p> Conclusions <p><i>Atg</i>5 affects protein expression and abundance in both the caput epididymis (segments 4–5) and cauda epididymal sperm, without statistically significant effects on sperm motility or male fertility. However, the potential impact of autophagy inhibition on sperm maturation may arise under challenging survival conditions by influencing the expression of sperm proteins.</p>