Fluoxetine, an antidepressant, has been indicated to elicit anti-cancer response in hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC) in vitro. However, anticancer effect and mechanism of fluoxetine in HCC and NSCLC in vivo still needs to be elucidated. In this study, we showed anticancer efficacy and inhibitory mechanism of fluoxetine on the tumor progression of HCC and NSCLC in vivo. Tumor growth was significantly inhibited with fluoxetine treatment in HCC and NSCLC in vivo. Fluoxetine obviously decreased expression of cell proliferative, anti-apoptotic, invasion-associated proteins including Cyclin-D1, survivin, vascular endothelial growth factor (VEGF), matrix metallopeptidase 9 (MMP-9) and urokinase-type plasminogen activator (uPA). Importantly, fluoxetine diminished the phosphorylation of NF-κB p65 which recognized as one of the critical transcription factors in tumor progression. Inhibition of AKT or extracellular signal-regulated kinases (ERK) phosphorylation was linked to NF-κB inactivation in NSCLC or HCC in vitro. Furthermore, expression of AKT or ERK phosphorylation was effectively attenuated by fluoxetine treatment in NSCLC or HCC in vivo. In addition, fluoxetine also triggered extrinsic/intrinsic apoptotic signaling by activating caspase-3, -8, and -9 in HCC and NSCLC. Our findings suggest that fluoxetine may represent as a promising adjuvant for patients with HCC or NSCLC. In conclude, the results also suggested the blockage of AKT/NF-κB or ERK/NF-κB activation and the induction of apoptosis are associated with fluoxetine-inhibited tumor progression of HCC or NSCLC in vivo.