Plant litter plays a key role in grassland biogeochemical cycles, yet the mechanisms through which short-term litter alterations regulate soil nutrient accumulation in desert steppes remain unclear. In a two-year field experiment with litter removal, control, and litter addition treatments, we examined how litter manipulation influences soil microclimate, plant growth, microbial community structure, extracellular enzyme activity, and soil nutrients. Compared with control, litter removal exhibited higher soil moisture but did not significantly affect plant biomass, height, density, or coverage in either year. By contrast, litter addition significantly increased those plant growth parameters in 2023, while maintaining soil moisture similar to the control in both years, suggesting that the moisture-conserving effect of the litter layer was offset by increased plant water uptake. Treatment effects on microbial communities and enzyme activities were stronger in 2023 than in 2024. Overall, litter addition increased soil dominant microbial phyla abundances, enhanced microbial alpha-diversities, and accelerated enzyme activities. Litter removal maintained similar values to the control for dominant microbial phyla abundances and microbial alpha-diversity, but tended to suppress enzyme activities. Correlation analysis revealed fungal taxa were positively correlated with soil moisture, and both fungal and prokaryotic abundances were positively correlated with plant biomass. Structural equation modeling confirmed that soil nutrient accumulation was directly influenced by prokaryotic community under litter removal, but positively driven by enzyme activities and fungal community under litter addition. Therefore, short-term litter addition can improve soil moisture and activate microbial-enzymatic processes in the desert steppe, which in turn promote soil nutrient accumulation.