Regeneration substrates may differ in water and nutrient availability, and that may lead to differ- ences in physiological traits among seedlings. This assumption was tested by investigating the effects of different regeneration substrates (fallen dead wood and soil) on physiological traits in seedlings of Rhododendron calophytum Franch. in a subalpine conifer forest, southwest China. The seedlings were divided into three groups according to their regener- ation substrates: moderately decayed dead wood (DWm); severely decayed dead wood (DWs) and soil (S). The results showed that there were significant differences in leaf mass per area (LMA), photosynthetic capacity, non-structural carbohydrate composition (NSC), leaf phosphorus (P) concentration, carbon isotope composition(δ 13 C) and ultrastructural morphol- ogy, not only between the seedlings regenerated in soil and on dead wood, but also between the seedlings regenerated on dead wood of different decaying stages. Overall, the photosynthetic capacity of the seedlings increased in the order of DWm <S < DWs, and the long-term water use efficiency (WUE) of the seedlings increased in the order of DWm <D Ws <S. Different nutrient and water availability in the substrates might be responsible for these differences. We suggest that severely decomposed fallen dead wood is the most suitable substrate for the regeneration of R. calophytum.