Altitude plays a crucial role in shaping the diversity and distribution of species in forest landscapes in mountainous regions. However, existing studies often lack comprehensive analysis of the intricate relationships among environmental factors, disturbances, and species diversity. This study aims to address this gap by thoroughly analyzing major species’ diversity, distribution, and regeneration status, along with their influencing factors, across different altitudinal gradients (1850 to 2350 m). Field surveys were conducted to examine the major species in different vegetation layers of the secondary Picea forest in Guandi Mountain. The key findings from the study include the following: (1) the study area recorded a total of nine species of adult trees, belonging to eight genera in five families, fourteen species of shrubs from eight genera in five families, and fifty-two herb species representing forty-eight genera in twenty-six families. Dominant species varied across layers, with Picea asperata Mast. and Larix principis-rupprechtii Mayr. dominating the arborous layer, Lonicera ferdinandii Franch. and Cotoneaster acutifolius Turcz. in the shrub layer, and Poa pratensis L. and Duchesnea indica (Andrews.) Focke in the herb layer. (2) Altitude significantly influenced species diversity, with the most pronounced effects observed in the shrub and herb layers. (3) Tree species regeneration varied with altitude, with Picea asperata exhibiting the highest regeneration performance, particularly in the high-altitude zone (2250–2350 m). Regeneration parameters were significantly correlated with species diversity indices in both tree and shrub layers. (4) Redundancy analysis revealed that, apart from altitude, cutting (p = 0.015) influenced the species diversity of the tree layer, while annual precipitation (p = 0.006) and temperature (p = 0.01) determined the diversity of the shrub layer. Grazing (p = 0.042) was identified as a determining factor for species diversity in the herb layer. Overall, these findings provide valuable insights into the sustainable management of forest ecosystems in the study area and offer practical guidance for forest management in similar mountainous landscapes worldwide.