High grade invasive bladder cancer is a leading cause of cancer deaths and treatment options are limited for this type of cancer. Recent studies have reported anticancer effects of valproic acid in many cancers and also in superficial bladder cancer. Acute valproic acid administration suppressed cell proliferation in a time- and dose-dependent manner in two muscle-invasive human bladder cancer cell lines (HTB5 and HTB9), with accompanying G1 phase cell cycle arrest. A significant decrease in colony formation ability and invasiveness was seen with valproic acid treatment though the effectiveness varied with cell type. Our results suggest a role for valproic acid in inhibiting growth and invasion of muscle-invasive bladder cancer.