Chronic exposure to nicotine significantly exacerbates periodontitis, a prevalent inflammatory disease, by inducing cellular processes such as autophagy and inflammation in gingival fibroblasts. Current therapies often fail to fully address these cellular alterations in smokers, highlighting a need for innovative therapeutic and regenerative approaches. This study explores the therapeutic potential of Platelet-Rich Plasma (PRP), a blood-derived product, to modulate nicotine-induced biological activities in primary gingival fibroblasts, particularly in the case of periodontitis in smokers. Gingival fibroblasts were treated with increasing concentrations of nicotine, which led to senescence and autophagy. Nicotine at high concentrations triggered cellular vacuolization, and a decrease in metabolism, viability and proliferation. Concomitant cell treatment with 10% PRP reversed nicotine effects and significantly increased cell migration potential. In Caenorhabditis elegans, PRP reduced the nicotine-induced autophagic activity. A screening of the gingival fibroblast secretome revealed a modulation of autophagy-related cytokines in response to nicotine and/or PRP. The findings demonstrate that PRP could effectively inhibit nicotine-induced autophagy in gingival fibroblasts, offering insights into its possible use as a therapeutic tool for managing periodontitis in smokers. The study underscores the potential of PRP in altering disease progression by modulating key cellular processes affected by smoking.