TSC1 is often mutated in bladder cancer. However the importance of this event in disease pathogenesis and its implications for therapy are uncertain. We used genomic sequencing to examine the involvement of TSC1 in bladder cancer, and signalling pathway analysis and small‐molecule screening to identify targeted therapeutic strategies in TSC1 mutant bladder cancer cell lines. TSC1 loss of heterozygosity was seen in 54% of bladder cancers. Two (4.9%) of these 41 bladder cancers had TSC1 mutations by exon‐based sequencing. Analysis of 27 bladder cancer cell lines demonstrated inactivating TSC1 mutations in three: RT‐4, HCV29, 97–1. Interestingly, only RT‐4 showed classic feedback inhibition of AKT, and was highly sensitive to treatment with mTOR inhibitors rapamycin and Torin1. 97–1 cells showed constitutive EGFR activation, and were highly sensitive to combined treatment with the mTOR inhibitor Torin1 and EGFR inhibitors Lapatinib or Afatinib. A BRAF missense mutation G469V was found in HCV29 cells, and AKT activation was dependent on BRAF, but independent of ERK. A kinase inhibitor screen of HCV29 cells showed strong growth inhibition by the Hsp90 inhibitor NVP‐AUY922, and we then found synergistic inhibitory effects of NVP‐AUY922 combined with either Torin1 or rapamycin on cell survival for both HCV29 and 97–1 cells. In aggregate, these findings indicate that there are highly variable mutation profiles and signalling pathway activation in TSC1‐mutant bladder cancer. Furthermore, combined Hsp90/mTOR inhibition is a promising therapeutic approach for TSC1 mutant bladder cancer. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.