This paper is the result of oil and gas exploration engineering. Objective It is necessary to study the pore structure characteristics of Niutitang Formation shale in Southeast Guizhou and its influence on shale gas enrichment, avoid unfavorable factors, and provide suggestions for shale gas exploration in Southeast Guizhou. Methods Multi-scale characterization of the shale micro-nano pore structure is carried out by means of focused ion beam scanning electron microscopy (FIB-SEM), FE-SEM, and nitrogen adsorption. Combined with the hydrocarbon generation and burial history of the Niutitang Formation shale in the study area, the relationship between tectonic movement and pore structure is studied, and its control effect on shale gas enrichment is analyzed. Results The Niutitang Formation shale mainly develops millimeter-scale micro-cracks, micron-scale pyrite intercrystalline pores and nano-scale organic pores, mainly ink bottle-shaped pores and slit pores with a flat plate structure, and the pore structure is complex. Combined with the results of FIB-SEM three-dimensional reconstruction, organic matter accounts for a relatively high proportion of shale, with a porosity of 0.04%-2.48% and a contribution rate of 14%-96% to the total porosity. The pyrite crystals co-existing with organic matter Pores are the main type of pores that communicate fractures, the shale gas in this area is dominated by undersaturated adsorbed gas, and the free gas content is low. Conclusions A large number of structural fractures in the Niutitang Formation shale have connected organic matter pores and pyrite intercrystalline pores, and changed the original pore structure, causing the in-situ accumulated shale gas to escape along the fractures, which is a key factor in the low gas content of the shale of the Niutitang Formation. Finding areas with moderate thermal evolution and well-preserved structures is a favorable direction for shale gas exploration on the periphery of paleo-uplifts.