AFAP1-AS1 is a long non-coding RNA that is associated with tumorigenesis and poor prognosis in a variety of cancers. We have been suggested that AFAP1-AS1 increases tumorigenesis in laryngeal carcinoma specifically by enhancing stemness and chemoresistance. We assessed AFAP1-AS1 expression in human laryngeal specimens, paired adjacent normal tissues and human HEp-2 cells. Indeed, we found not only that AFAP1-AS1 was up-regulated in laryngeal carcinoma specimens and cells, but also that stemness-associated genes were overexpressed. Silencing of AFAP1-AS1 promoted HEp-2 cell chemoresistance under cisplatin treatment. Expression of AFAP1-AS1 was increased in drug-resistant Hep-2 cells. We then probed the mechanism of AFAP1-AS1 activity and determined that miR-320a was a potential molecular target of AFAP1-AS1. Luciferase reporter and qRT-PCR assays of AFAP1-AS1 and miR-320a levels in human specimens and cell cultures indicated that AFAP1-AS1 negatively regulates miR-320a. To discover the molecular mechanism of miR-320a, we again used the DIANA Tools algorithm to predict its genetic target, RBPJ. After cloning the 3'-untranslated regions (3'-UTR) of RBPJ into a luciferase reporter, we determined that miR-320a did in fact reduce RBPJ mRNA and protein levels. Ultimately, we determined that AFAP1-AS1 increases RBPJ expression by negatively regulating miR-320a and RBPJ overexpression rescues stemness and chemoresistance inhibited by AFAP1-AS1 silencing. Taken together, these results suggest that AFAP1-AS1 can serve as a prognostic biomarker in laryngeal carcinoma and that miR-320a has the potential to improve standard therapeutic approaches to the disease, especially for cases in which cancer cell stemness and drug resistance present significant barriers to effective treatment.