Interferon beta (IFN beta) is transiently expressed in response to viral infections and widely used to treat relapsing-remitting multiple sclerosis (MS). We introduced mutations in the IFN beta gene (in the 27th and 101st codons and in the Kozak sequence, and also deletion of 3' and 5' unstable, untranslated region, UTR) with the aim of increasing the expression of IFN beta. Computational analyses of mutant and wild-type RNAs and proteins of IFN beta by RNAfold, ASAView, HOPE and Ramachandran plot, and iStable web servers showed that the mutations could decrease RNA stability, protein solvent accessibility, and protein stability but could not change correct folding. Two recombinant IFN beta 101 and IFN beta 101+27 constructs were designed by site-directed mutagenesis. The wild-type IFN beta gene also was used as a control. In vitro experiments by quantitative real-time PCR, dot blot, SDS-PAGE, and Western blot assays showed an increased expression level of recombinant IFN beta s. 79.9-fold, 99.7-fold, and 190-fold elevations in the mRNA expression of IFN beta w, IFN beta 101, and IFN 101+27 were seen, respectively, in comparison with the endogenous IFN beta mRNA in untransfected HEK293T cells. The IFN beta mRNA expression was increased 2.38-fold and 1.25-fold for 101+27 and 101 mutated forms, respectively, in comparison with the IFN beta wild-type construct. An elevation in IFN beta protein production was also clearly detected in the transfected HEK293T cell containing recombinant IFN 101 and IFN beta 101+27 constructs. Finally, these directed mutations in the IFN beta gene successfully elevated protein and mRNA production but in silico analyses of mutant mRNAs showed decreased mRNA stability, unlike previous studies, in comparison with the wild-type mRNA.