<p><i>Pinus massoniana</i> (Masson pine) is an ecologically and economically important conifer distributed throughout central and southern China and widely planted for its timber and resin, according to previous studies. However, its recalcitrance to genetic transformation under in vitro culture conditions has limited progress in functional genomics research. To address this constraint, we established an efficient platform for protoplast isolation and polyethylene glycol (PEG)-mediated transient transformation in <i>P. massoniana</i>. Optimized enzymatic digestion, utilizing 2% cellulase (needles) or 1% cellulase (stems) combined with 0.125% pectinase, yielded protoplasts at densities &gt; 1 × 10⁷ protoplasts/mL with &gt;90% viability. Employing GFP as a reporter, optimized PEG4000-mediated transfection (30%, 15 min for needles; 15%, 45 min for stems) achieved efficiencies exceeding 60%. Expression and subcellular localization of both an exogenous gene (<i>PeWOX5</i>) and an endogenous gene (<i>PmbHLH44</i>) were successfully demonstrated using this system. Collectively, this platform provides a robust tool for rapid gene function analysis in <i>P. massoniana</i> and establishes a foundational methodology for advancing molecular research in coniferous species.</p>