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Guizhou Province is located in the karst mountain regions of Southwest China, where the ecological environment is extremely fragile and particularly sensitive to human activities. Therefore, understanding the changing characteristics and driving factors of human activity in recent decades is urgent. In this study, least squares, correlation analysis, spatial autocorrelation analysis, and GeoDetector model were used. Based on a large collection of nightlight, topography, and meteorological data, combined with geographical information technology, we investigated the spatial distribution, intensity change, and degree of impact of human activity from 2000 to 2020. The results showed that during the study period, human activities intensity and range were expanding. The human activity areas expanded from 15,963 to 86,923 km2 at an increasing rate of 4,279.2 km2/a, which was 1,118.4 km2/a from 2000 to 2010 and 6,375.3 km2/a from 2010 to 2020. The human activity intensity increased from 0.009 to 0.018, with an increasing rate of 0.0006/a, which was 0.00028/a from 2000 to 2010 and 0.00073/a from 2010 to 2020. The rate of increase in the human activity intensity and the rate of expansion in the human activity range from 2010–2020 were significantly higher than that from 2000–2010, becoming more notable (p ≤ 0.01), especially after 2012. With the expansion of human activities, the center of gravity of human activity moved towards the northeast by 20.71 km. The human activities were primarily distributed in areas with the gentlest slopes (6–15°), middle and low altitudes (489–1,982 m), suitable temperatures (12.36–17.74°C), and abundant precipitation (1,001.99–1,276.99 mm). The research results using the GeoDetector model indicate that slope had the greatest impact on human activities with a q value of 0.1338; precipitation, elevation, and temperature had q values of 0.0626, 0.0253, and 0.0136, respectively. The combined impact between the precipitation and slope was the greatest with a q value of 0.1803. In Guizhou Province, under policy guidance, human activities that promoted vegetation change accounted for 79.60%. This study attempts to enhance sustainable development and provides valuable information on the environmental protection of karst mountain regions.