Long noncoding RNAs (lncRNAs) have been reported to exhibit a crucial regulatory role in tumor progression, including cholangiocarcinoma (CCA). As a promising lncRNA, proteasome 20S subunit alpha 3 antisense RNA 1 (PSMA3-AS1) is involved in development of various tumors. However, the role and function of PSMA3-AS1 in CCA remain unclear. The aim of this study is to examine the expression, function, mechanism, and clinical significance of PSMA3-AS1 in CCA development. By TCGA database analysis, we found that PSMA3-AS1 was overexpressed in CCA. Consistent with the TCGA analysis, PSMA3-AS1 was significantly overexpressed in CCA tissues and cells by RT-qPCR. Upregulated PSMA3-AS1 was related to lymph node invasion, advanced TNM stage and poor survival, and was an independent risk factor of prognosis for CCA patients. Functionally, CCK-8, EdU and colony formation assays confirmed that upregulated PSMA3-AS1 promoted CCA cell proliferation, whereas downregulated PSMA3-AS1 inhibited proliferation. This result was further confirmed by subcutaneous tumor formation in nude mice. Wound healing and transwell assays confirmed that increased PSMA3-AS1 promoted CCA cell migration and invasion, whereas decreased PSMA3-AS1 inhibited these biological phenotypes. In addition, PSMA3-AS1 promoted the EMT process of CCA by downregulating E-cadherin and upregulating N-cadherin and vimentin. Mechanistically, transcription factor PAX5 bound to the promoter region of PSMA3-AS1 and promoted its transcription. Simultaneously, PSMA3-AS1 primarily localized in the cytoplasm could competitively bind miR-376a-3p to upregulate LAMC1, thereby accelerating CCA progression. This study uncovers that PSMA3-AS1 functions as a cancer-promoting gene in CCA, and PAX5/PSMA3-AS1/miR-376a-3p/LAMC1 axis plays a vital role in CCA development.