Osteoarthritis (OA) is an age-related disease marked by synovial inflammation and cartilage destruction arising from synovitis, joint swelling and pain. OA therapy that targets the synovium is a promising strategy for mitigating the symptoms and disease progression. Altered activity of the transforming growth factor-β1 isoform (TGF-β1) during aging underlies OA progression. Notably, aberrant forkhead box class O 3 (FOXO3) activity is implicated in the pathogenesis of various age-related diseases, including OA. This study explored the interaction and cross-talk of TGF-β1 and FOXO3 in human osteoarthritis synovial fibroblasts (OASFs). TGF-β1 stimulated FOXO3 synthesis in OASFs, which was mitigated by blocking adenosine monophosphate-activated protein kinase (AMPK) and p38 activity. TGF-β1 also inhibited the expression of miR-92a, which suppresses FOXO3 transcription. The suppression of miR-92a was effectively reversed with the blockade of the AMPK and p38 pathways. Our study showed that TGF-β1 promotes anti-inflammatory FOXO3 expression by stimulating the phosphorylation of AMPK and p38 and suppressing the downstream expression of miR-92a. These results may help to clarify OA pathogenesis and lead to better targeted treatment.