Spatio-temporal variation analysis of water resources vulnerability can provide theoretical support for the planning and management of water resources and the mitigation of water resources vulnerability. Based on the pressure-state-response(PSR) model, the water resources vulnerability in Guizhou Province from 2004 to 2017 was analyzed using a combined weighting method. The results showed that the water resources vulnerability index of Guizhou Province showed a downward trend in fluctuation from 2004 to 2017. The average vulnerability index of the whole province was 0.486, which was classified as moderately vulnerable. The calculation results based on the PSR model were pressure index> state index> response index. The vulnerability index presented obvious spatial differences, and the water resources vulnerability problems faced by the cities (states) in this province remained severe. The main reason of the over high vulnerability index in some areas of the province was caused by the high pressure on water resources, which resulted in poor water environment. Total waste water discharge, agricultural fertilizer application, rainfall and total water resources were important factors affecting the vulnerability of the province’s water resources. In general, the vulnerability of water resources in Guizhou was moderately vulnerable with obvious spatial differences which posed a great threat to the water resources. Therefore, studying the vulnerability of water resources in Guizhou Province can provide a theoretical basis for the protection and management of water resources in Guizhou, and also serve as a reference on exploring the vulnerability of water resources in karst areas.