Antisense long noncoding RNAs (lncRNAs) play important roles in regulating the expression of coding genes in post-transcriptional level. However, detailed expression profile of lncRNAs and functions of antisense lncRNAs in bladder cancer remains unclear. To investigate regulation of lncRNAs in bladder cancer and demonstrate their functions, we performed lncRNAs microarray analysis in 3 paired bladder cancer tissues. Further molecular assays were conducted to determine the potential role of identified antisense lncRNA MDC1-AS. As a result, a series of lncRNAs were differentially expressed in bladder cancer tissues in microarray screen. In a larger size of samples validation, we found that the expression levels of MDC1-AS and MDC1 was down-regulated in bladder cancer. After over-expression of MDC1-AS, increased levels of MDC1 were observed in bladder cancer cells. We also found a remarkably inhibitory role of antisense lncRNA MDC1-AS on malignant cell behaviors in bladder cancer cells EJ and T24. Subsequently, knockdown of MDC1 revealed that suppressing role of MDC1-AS was attributed to up-regulation of MDC1. In summary, we have identified a novel antisense lncRNA MDC1-AS, which may participate in bladder cancer through up-regulation of its antisense tumor-suppressing gene MDC1. Further studies should be conducted to demonstrate detailed mechanism of our findings.