Micro-RNAs are dysregulated in medullary thyroid carcinoma (MTC) and preliminary studies have shown that miRNAs may enact a therapeutic effect through changes in autophagic flux. Our aim was to study the in vitro effect of miR-9-3p on MTC cell viability, autophagy and to investigate the mRNA autophagy gene profile of sporadic versus hereditary MTC. The therapeutic role of miR-9-3p was investigated in vitro using human MTC cell lines (TT and MZ-CRC-1 cells), cell viability assays, and functional mechanism studies with a focus on cell cycle, apoptosis, and autophagy. Post-miR-9-3p transfection mRNA profiling of cell lines was performed using a customized, quantitative RT-PCR gene array card. This card was also run on clinical tumor samples (sporadic: n = 6; hereditary: n = 6) and correlated with clinical data. Mir-9-3p transfection resulted in reduced in vitro cell viability; an effect mediated through autophagy inhibition. This was accompanied by evidence of G2 arrest in the TT cell line and increased apoptosis in both cell lines. Atg5 was validated as a predicted miR-9-3p mRNA target in TT cells. Post-miR-9-3p transfection array studies showed a significant global decline in autophagy gene expression (most notably in PIK3C3, mTOR, and LAMP-1). Autophagy gene mRNAs were generally overexpressed in sporadic (vs. hereditary MTC) and Beclin-1 overexpression was shown to correlate with residual disease. Autophagy is a tumor cell survival mechanism in MTC that when disabled, is of therapeutic advantage. Beclin-1 expression may be a useful prognostic biomarker of aggressive disease.