Increasing evidences show that ethylene-responsive factor (ERF) proteins regulate plant stress response and the interaction of different stress responsive pathways through interacting with different cis-acting elements, even other transcription factors. Here, we report a transcriptional activator TSRF1, which was previously demonstrated to regulate plant resistance to Ralstonia solanacearum, reversely regulates pathogen resistance and osmotic stress tolerance in tobacco. Sequence analysis revealed that TSRF1 contains a putative transcriptional activation domain. Using yeast two hybrid system we evidenced that this activation domain is essential for activating the expression of reporter gene. To confirm the broad-spectrum pathogen resistance of TSRF1 we observed that over-expressing TSRF1 enhances the resistance to Pseudomonas syringae and Botrytis cinerea in both tobacco and tomato plants, but RNA interference of TSRF1 in tomato plants decreases the resistance to these pathogens, unraveling the positive regulation of TSRF1 in plant pathogen infections. The expression of TSRF1 in response to NaCl and mannitol suggests the possible functions of TSRF1 in osmotic stress responses, but the physiological tests indicate that expressing TSRF1 in tobaccos decreases tolerance to NaCl or mannitol during germination and seedling root development, and this result was consistent with PEG6000 treatment with mature tobacco seedlings, indicating the negative modulation of TSRF1 in osmotic stress response. Therefore, our research reveals that transcriptional activator TSRF1 reversely regulates plant pathogen resistance and osmotic stress response.