Gaps by thinning can have different microclimatic environments compared to surrounding areas, depending on the size of the gap. In addition, gaps can play important roles in biological dynamics, nutrient cycling, and seedling regeneration. The impacts of gap size on soil microbial communities and enzyme activities in different soil layers in Chinese pine plantations are not well understood. Here, we created gaps of 45 m2 (small, G1), 100 m2 (medium, G2), and 190 m2 (large, G3) by thinning unhealthy trees in an aged (i.e., 50 years old) monoculture Chinese pine plantation in 2010. Soil samples were collected in 2015. The total, bacterial, Gram-positive (G+), and Gram-negative (G−) phospholipid fatty acid (PLFA) profiles were highest in medium gaps in both the organic and mineral layers. These indicesdecreased sharply as gap size increased to 190 m2, and each of the detected enzyme activities demonstrated the same trend. Under all the gap size managements, abundances of microbial PLFAs and enzyme activities in the organic layers were higher than in the mineral layers. The soil layer was found to have a stronger influence on soil microbial communities than gap size. Redundancy analysis (RDA) based on the three systems with different gap sizes showed that undergrowth coverage, diversity, soil total nitrogen (TN), total organic carbon (TOC), and available phosphorus (AT) significantly affected soil microbial communities. Our findings highlighted that the effect of gap size on soil microenvironment is valuable information for assessing soil fertility. Medium gaps (i.e., 100 m2) have higher microbial PLFAs, enzyme activity, and soil nutrient availability. These medium gaps are considered favorable for soil microbial communities and fertility studied in a Chinese pine plantation managed on the Loess Plateau.