Long noncoding RNAs (lncRNAs) act as key regulators controlling complex cellular behaviors in nonsmall cell lung cancer (NSCLC). We investigated the expression of lncRNA PRRT3 antisense RNA 1 (PRRT3-AS1) in paired samples of NSCLC and adjacent normal tissues from a patient cohort in our hospital using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and found that it was significantly higher in NSCLC tissue than in normal tissue, consistent with The Cancer Genome Atlas database. Furthermore, functional investigation revealed that lncRNA PRRT3-AS1 depletion inhibited NSCLC-cell proliferation, colony formation, invasion, and migration, whereas its overexpression exerted the opposite effects. Moreover, PRRT3-AS1 knockdown suppressedin vivoNSCLC growth. Investigation of downstream mechanisms using RNA immunoprecipitation and luciferase reporter assay revealed that lncRNA PRRT3-AS1 acted as a competing endogenous RNA by adsorbing microRNA-507 (miR-507) and enhanced the expression of its target gene, homeobox B5 (HOXB5), in NSCLC. Furthermore, miR-507 downregulation or HOXB5 upregulation eliminated the cancer-inhibiting effects of lncRNA PRRT3-AS1 depletion in NSCLC cells. To conclude, the lncRNA PRRT3-AS1/miR-507/HOXB5 pathway acts as a promoter of malignant characteristics in NSCLC, and this newly identified competing endogenous RNA pathway may be an effective diagnostic, prognostic, and therapeutic target in NSCLC.