Bladder cancer (BC) is the ninth most malignant tumor worldwide. Some BC patients will develop muscle‐invasive BC (MIBC), which has a 5‐year survival rate of approximately 60% due to metastasis. As such, there is an urgent need for novel therapeutic and diagnostic targets for MIBC. Analysis of novel antitumor microRNA (miRNA)‐mediated cancer networks is an effective strategy for exploring therapeutic targets and prognostic markers in cancers. Our previous miRNA analysis revealed that miR‐140‐5p acts as an antitumor miRNA in BC cells. Here, we investigated miR‐140‐5p regulation of BC molecular pathogenesis. Procollagen‐lysine, 2‐oxoglutarate 5‐dioxygenase 1 (PLOD1) was found to be directly regulated by miR‐140‐5p, and aberrant expression of PLOD1 was observed in BC clinical specimens. High PLOD1 expression was significantly associated with a poor prognosis (disease‐free survival: P = 0.0204; overall survival: P = 0.000174). Multivariate analysis showed PLOD1 expression to be an independent prognostic factor in BC patients (hazard ratio = 1.51, P = 0.0099). Furthermore, downregulation of PLOD1 by siRNAs and a specific inhibitor significantly decreased BC cell aggressiveness. Aberrant expression of PLOD1 was closely associated with BC pathogenesis. In summary, the present study showed that PLOD1 may be a potential prognostic marker and therapeutic target for BC.