ObjectiveWe aimed to characterize the expression patterns, gene targets, and functional effects of miR-335-5p and miR-335-3p among seven primary human knee and hip osteoarthritic tissue types.MethodsWe collected synovial fluid, subchondral bone, articular cartilage, synovium, meniscus/labrum, infrapatellar/acetabular fat, anterior cruciate ligament/ligamentum teres, and vastus medialis oblique/quadratus femoris muscle (n= 7–20) from surgical patients with early- or late-stage osteoarthritis (OA) and quantified miR-335-5p and miR-335-3p expression by real-time PCR. Predicted gene targets were measured in knee OA infrapatellar fat following miRNA inhibitor transfection (n= 3), and prioritized gene targets were validated following miRNA inhibitor and mimic transfection (n= 6). Following pathway analyses, we performed Oil-Red-O staining to assess changes in total lipid content in infrapatellar fat.ResultsShowing a 227-fold increase in knee OA infrapatellar fat (the highest expressing tissue) versus meniscus (the lowest expressing tissue), miR-335-5p was more abundant than miR-335-3p (92-fold increase). MiR-335-5p showed higher expression across knee tissues versus hip tissues, and in late-stage versus early-stage knee OA fat. Exploring candidate genes,VCAM1andMMP13were identified as putative direct targets of miR-335-5p and miR-335-3p, respectively, showing downregulation with miRNA mimic transfection. Exploring candidate pathways, predicted miR-335-5p gene targets were enriched in a canonical adipogenesis network (p= 2.1e − 5). Modulation of miR-335-5p in late-stage knee OA fat showed an inverse relationship to total lipid content.ConclusionOur data suggest both miR-335-5p and miR-335-3p regulate gene targets in late-stage knee OA infrapatellar fat, though miR-335-5p appears to be more prominent, with tissue-, joint-, and stage-specific effects.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13075-023-03088-6.