As an innovative class of promising cancer therapeutic, oncolytic adenovirus has been commonly used recently due to its ability to infect and lyse cancer cells specifically, while ideally leaving normal cells unharmed. A potential advantage of oncolytic adenoviral vectors over conventional antitumor agents is that viral replication in the tumor will amplify the input virus dose, leading to spread of the virus throughout the tumor. To observe the inhibiting effect of oncolytic adenovirus on the lung cancer cell growth in combination with hyperthermia and to determine whether it can improve the expression levels of therapeutic gene delivered by replication-deficient adenovirus, an oncolytic adenovirus named Ad-HSP70p-E1A was constructed by employing the human HSP70 gene promoter to drive the expression of adenovirus E1A gene. The experimental results show that in vitro, Ad-HSP70p-E1A can replicate in lung carcinoma A549 cell line and destroy those cancer cells to some extent. While combined with hyperthermia, the number of replicative virions released from lung carcinoma increased about 2～10 times and the oncolytic effect on lung carcinoma A549 cell line increased 5 times at least. In addition, when combined with oncolytic adenovirus Ad-HSP70p-E1A, replication-deficient adenoviruses such as AdGFP, Ad-CMV-hGMCSF and Ad-CMV-mIL12 at the moi of 10 are expected to lead to the expression level of therapeutic gene at higher levels. The green fluorescence protein increased 76.64 times, while cytokines GMCSF and IL12 increased 5 times and 7 times respectively.