MicroRNAs (miRs) are small non-coding RNAs that regulate the translation of many genes in normal and cancer cells where they are frequently dysregulated promoting tumor progression. Several studies have illustrated the potential of manipulating miR expression in cancer research and therapy. The aim of the present study was to investigate expression patterns of a panel of miRs in head and neck squamous cell carcinoma (HNSCC) shown to be relevant in other carcinomas and to elucidate their role if dysregulated. We performed analysis of miR‑21, -200c, -138-1, -138-2, -25 and -34 expression by qRT-PCR in 6 HNSCC cell lines and computerized search for genetic targets of dysregulated miRNA-21 (miR‑21). Lipofection of mock and anti-miR-21 and determination of expression efficiencies and final programmed cell death 4 (PDCD4) expression were carried out by luciferase assay and western blotting. MTT assay was used to measure cell proliferation and flow cytometry was performed for cell cycle analysis. Expression of miR-21 was most prominently upregulated in the HNSCC cell lines, particularly in UM-SCC11B (6.45±0.25-fold, P<0.05) and UM-SCC9 (4.35±0.22-fold, P<0.05) as compared to primary epidermal keratinocytes used as control. The expression levels of the other miRs showed no difference except for miR-34 and -138-1 each in one cell line. Subsequent transfection of precursor miR-21 stimulated proliferation while anti-miR-21 inhibited proliferation of both cell lines. PDCD4 was identified with software designed for this purpose as potential target gene of miR-21. Subsequently, its role in HNSCC lines was experimentally confirmed by regulation of PDCD4 transfecting miR-21 mimics and anti-miR-21. Finally, we showed that PDCD4 is negatively regulated by miR-21 at the post-transcriptional level via binding to the 3'-untranslated region of PDCD4 mRNA. A role of upregulated miR-21 and reduced PDCD4 stimulating the proliferation was demonstrated in HNSCC lines and, in turn, transfection of anti-miR-21 upregulating PDCD4 reduced the cellular division rate. We explored miR-21 and PDCD4 expression as markers of progression and prognosis and for a potential translational value in the development of agents slowing growth of HNSCC and other carcinomas useful in palliative therapy or as a component of multi-modality treatments.