Minicircle DNA (mcDNA) has been suggested as a vanguard technology for gene therapy, consisting of a nonviral DNA vector devoid of prokaryotic sequences. Unlike conventional plasmid DNA (pDNA), this small vector is able to sustain high expression rates throughout time. Thus, this work describes the construction, production, and purification of mcDNA- and its precursor parental plasmid (PP)- for a comparative study of both DNA vectors in the growth suppression of human papillomavirus (HPV)-18-infected cervical cancer cells. First, live cell imaging and fluorescence microscopy studies allowed to understand that mcDNA- vector was able to enter cell nuclei more rapidly than PP- vector, leading to a transfection efficiency of 68% against 34%, respectively. Then, transcripts and protein expression assessment revealed that both vectors were able to induce transcription and the target protein expression. However, the mcDNA- vector performance stood out, by demonstrating higher expression levels (91.65 ± 2.82 U/mL vs. 74.75 ± 4.44 U/mL). After assuring the safety of both vectors by viability studies, such potential was confirmed by proliferation and apoptosis assays. These studies confirmed the mcDNA- vector function toward cell cycle arrest and apoptosis in HPV-18-infected cervical cancer cells. Altogether, these results suggest that the mcDNA vector has a more promising and efficient role as a DNA vector than conventional pDNA, opening new investigation lines for cervical cancer treatment in the future.