Straw return is a key method to regulate soil carbon (C) by microbial utilization accumulating and CO2 releasing, which mainly depends on microbial metabolism processes. The present knowledge of the mode and mechanism of soil CO2 emission under straw return is limited. We established four straw return treatments, including straw shallow return (SS, 0–20 cm), straw subsoil burial (SD, 20–40 cm), straw mulching (SM), and control with no straw (NS). Microbial metabolic limitation and the abundance of C degradation functional genes were quantified using enzyme stoichiometry and high-throughput quantitative PCR-based chip technology, respectively. The results revealed that the addition of straw inputs resulted in an increase in soil nutrient contents and soil CO2 emissions. The cumulative CO2 emissions of SS and SM treatments were 21,013.53 and 20,819.26 kg hm−2, respectively, which significantly increased by 4.70 % and 3.73 % compared with SD treatment. Compared with SM and SD treatments, microbial C and P limitations of SS treatment were significantly enhanced, increasing by 1.15 %–1.20 % and 2.73 %–1.21 %, respectively. Straw return increased the absolute abundance of genes engaged in decomposing diverse C source compounds, although with a reduction in microbial C use efficiency. The absolute abundance of amyA, apu, sga, abfA, xylA, and manB labile C degradation genes was higher in SM than in SD treatment, increasing by 1.11 %–25.88 %. On the contrary, the absolute abundance of glx and lig recalcitrant C degradation genes was lower in SM than in SD treatment, reducing by 2.96 %–3.68 %. Straw return directly impacts soil nutrient contents and indirectly influences microbial biomass carbon (MBC), nutrient limitation, and C degradation genes, as revealed by the partial least squares pathway model, ultimately leading to changes in soil CO2 emissions. This finding should contribute to the enhancement of soil C retention and the selection of appropriate methods for straw incorporation in tillage fields.