Physalis pubescens L. plant produces nutritious and healthy fruits, called husk tomato or hairy ground cherry. However, its bioactive components are largely unknown. Four new withanolide steroids (1-4) together with one known withanolide (5) were isolated from the extract of P. pubescens L. and their chemical structures were established by extensive spectroscopic analyses. Compounds 1, 3 and 5 showed potent growth inhibitory effects against four human renal cell carcinoma (RCC) cell lines (i.e. 786-O, A-498, Caki-2 and ACHN). Among them, compound 1 was the most potent one with IC50s ranged from 0.30 to 0.77 μM. Further experiment showed that 1 sensitized human RCC cells 786-O to the tumor necrosis factor related apoptosis ligand (TRAIL)-induced apoptosis and increased the expression of C/EBP-homologous protein (CHOP) and death receptor-5 (DR5), leading to activation of the DR5 and caspase-8/3 mediated apoptosis pathway. Molecular docking analysis revealed that compound 1 could bind stably to the TRAIL/DR5 complex through hydrogen bonds. These results suggest that the new withanolide (1) is a lead anti-cancer compound existing in P. pubescens L. and deserves further investigation for RCC prevention and treatment.