Interleukin (IL)-7 acts via the IL-7 receptor in metastatic tumor progression in prostate cancer (PC). The current study aimed to evaluate thymoquinone (Tq), an active constituent from Nigella sativa against IL-7–driven tumor progression and metastatic invasion in PC cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the proliferation of PC cells. Enzyme-linked immunosorbent assay was used to detect the expression of IL-7 and matrix metalloproteinases (MMPs). Tumor-cell transendothelial, scratch wound and cell scatter assays were performed to mimic metastasis. Western immunoblotting was used to measure the level of proteins. Tq effectively controlled the proliferation of DU-145, PC-3, and LNCaP cells with GI50 of 10.18, 12.40, and 16.78 µM, respectively. IL-7 and IL-7R were natively expressed in all PC types, while maximal expression was detected in DU-145. IL-7 promoted metastatic events, such as transendothelial migration, cell scatter, and cell invasion of DU-145 cells in a dose-dependent manner that was inhibited by Tq. Furthermore, Tq also downregulated p-Akt and NF-κB in DU-145 cells induced by IL-7 antibody and reduced the levels of MMP-3 and MMP-7 in these cells in a dose-dependent manner. Collectively, Tq has excellent efficacy in controlling tumor progression, migration, and invasion of DU-145 cells that were driven by the activation of MMPs through IL-7/Akt/NF-κB signaling.