The effects of environmental factors, such as soil moisture (SW), air temperature (T), air relative humidity (RH) and photosynthetic active radiation (PAR) on stomatal conductance (Gs) and total hydraulic conductance of soil—plant system (Kt) under steady flow condition were analyzed, and regulation effects of these impact factors on plant transpiration were investigated. Greenhouse cucumber was used as the experimental material, based on the design of composite quadratic orthogonal regressive rotation where the artificial climate box was adopted, the main effects, single effects and marginal effects of four environmental impact factors on Gs and Kt were interpreted. The results showed that all environmental factors exerted positive effect on Gs and Kt, interaction effect was also found between SW and PAR, T and RH on Gs and Kt. The single effect of RH can be described by a parabolic function and those of other impact factors were described as linear function, which were increased with increase of Gs and Kt. The marginal effects of each impact factor on Gs and Kt showed that RH was the main regulating path for Gs, and all the impact factors except PAR efficiently regulated Kt. The regulating effects of the four environmental factors, Gs and Kt on transpiration were analyzed with paths and correlation analyses. The results showed that T, PAR and Gs mainly exerted indirect positive effect on transpiration bystrengthening Kt, which was followed by the direct positive effect on transpiration. SW mainly exerted indirect positive effect on transpiration by strengthening both Kt and Gs. RH exerted direct negative effect, while its main effect path was exerting indirect positive effect on transpiration by promoting Gs and Kt. Both Gs and Kt were remarkably responded to the four environmental impact factors and interactively regulated the transpiration synergistically.