Bioactive gibberellins (GAs) are a type of important plant growth regulators, which play the key roles in multiple processes, such as seed germination, leaf expansion, flowering, fruit bearing, and stem development. Its biosynthesis is regulated by a variety of enzymes including gibberellin 3-oxidase that is a key rate-limiting enzyme. In Arabidopsis, gibberellin 3-oxidase consists of four members, of which AtGA3OX1 and AtGA3OX2 are highly expressed in stems, suggesting the potential roles in the stem development played by the two genes. To date, there are few studies on AtGA3OX1 and AtGA3OX2 regulating secondary wall thickening in stems. In this study, we used the atga3ox1atga3ox2 double mutant as the materials to study the effects of AtGA3OX1 and AtGA3OX2 genes on secondary wall thickening in stems. The results indicated that simulations repression of AtGA3OX1 and AtGA3OX2 genes resulted in significantly reduction of secondary wall thickening of fiber cells, but not that of vessel cells. Three main components (cellulose, hemicelluloses, and lignin) were also dramatically suppressed in the double mutants. qRT-PCR analysis demonstrated that the expressions of secondary wall biosynthetic genes and the associated transcription factors were obviously affected in AtGA3OX1 and AtGA3OX2 double mutant. Therefore, we presume that Arabidopsis AtGA3OX1 and AtGA3OX2 genes might activate the expression of these transcription factors, thus regulate secondary wall thickening in stems. Together, our results provide a theoretical basis for enhancing the lodging resistance of food crops and improving the biomass of energy plants by genetically engineering Arabidopsis AtGA3OX homologs.