The inhibitor of DNA-binding/differentiation 3 (Id3) protein is a helix-loop-helix transcription factor and may have an important role in cell proliferation and differentiation. This study was to evaluate the effects of upregulation of Id3 in human lung adenocarcinoma cells on proliferation, apoptosis, mobility and tumorigenicity. Short interference RNA suppression of Id3 (miRId3) in A549 cells was used to investigate the functional role(s) of Id3. Next, we used in vitro wound-healing assay and trans-well assay to study the effects of overexpressed Id3 on migration and invasion of A549 cells. Furthermore, to explore the influence of overexpressed Id3 on in vivo tumorigenesis, adenoviruses containing Id3 gene (Ad-Id3) and empty vector (Ad-LacZ) were generated. Co-transfection of pcDNA/miRId3 and pEGFP/Id3 into A549 cells reversed the Id3-induced cell proliferation inhibition and apoptosis. Upon Id3 transfection, A549 cells displayed decreased migratory and invasive capabilities, however, co-transfection of miRId3 and Id3 into A549 cells reversed the Id3-induced inhibitions of migratory and invasive capabilities. Three groups of nude mice were inoculated with Ad-LacZ, Ad-Id3 transfectants and untransfected A549 cells, respectively. Twenty-eight days after inoculation, tumors induced by Ad-Id3 transfectants grew much more slowly compared with Ad-LacZ transfectants and control group. This study provides for the first time both in vitro and in vivo proofs that forced expression of Id3 in lung adenocarcinoma cells reduces tumor growth rate and may be a potential target for tumor suppression.