Background Gut microbiota play a vital role in nutrient metabolism, immune regulation, and host homeostasis. However, the role of sex differences in shaping the gut microbiota of plateau zokors ( Eospalax baileyi ) remains unclear. The present study aims to explore how sex influences the composition, function, and assembly processes of the gut microbiota in plateau zokors. Methods In this study, we performed Illumina 16S rRNA (V3–V4) sequencing on 15 gastrointestinal samples to assess sex-related differences in gut bacterial diversity, function, and community assembly. Results No significant differences were observed in species richness or diversity between males and females; however, the gut microbial community structures differed significantly by sex ( p < 0.01). At the phylum level, both sexes shared dominant phyla, including Firmicutes, Desulfobacterota, and Bacteroidota. Across both the phylum and genus levels, males and females shared the same dominant taxa, yet their relative abundances exhibited clear sex-specific differences. PICRUSt-based functional prediction indicated that the gut microbiota were mainly associated with energy metabolism, DNA repair, and cellular defense. Significant sex-related differences were detected in metabolic functions ( p < 0.05), with males showing higher carbohydrate metabolism ( p < 0.05), while females exhibited stronger xenobiotic biodegradation and metabolism ( p < 0.05). Neutral community model (NCM) analysis showed that males (Nm = 228.21) had higher Nm values than females (Nm = 213.44), indicating greater microbial dispersal among males. Standardized neutrality score (NST) values (<0.5) indicated that deterministic processes predominantly governed community assembly in both sexes, with males exhibiting significantly lower values than females ( p < 0.001). iCAMP analysis further revealed that drift and dispersal limitation were the primary assembly processes, with significant sex-related differences ( p < 0.001). Conclusion Sex differences markedly influence gut microbial structure, functions, and assembly processes in plateau zokors, offering new insights into the adaptive evolution of this species in cold, hypoxic environments.