The objective of this study was to evaluate the performance of Lactobacillus acidophilus cells as a novel encapsulating carrier for fisetin via osmoporation. Initially, the effects of osmotic pressure and initial fisetin concentration on the performance of the osmoporation process were evaluated. The best results were achieved when 15 MPa was applied, while the maximum loading capacity was reached when fisetin concentration of 2.0 mg·mL-1 was used. For these conditions, the cell viability, encapsulation efficiency (EE), and encapsulated fisetin content (EF) were 72%, 28%, and 0.990 mg, respectively. Further, the encapsulation was confirmed by Fourier transform-infrared (FT-IR), differential scanning calorimetry (DSC), and X-ray diffraction (XRD) analysis. DSC thermograms revealed an increase of 40 °C in the melting point of fisetin after encapsulation. In addition, the enhancement of fisetin bioaccessibility by osmoporated biocapsules is shown for the first time in the literature. When the fisetin biocapsules were subjected to in vitro gastrointestinal digestion, 99.6% of the encapsulated content were retained through the gastric stage and 45.5% were released during the intestinal stage, despite no active cells were detected during simulated digestion. These results suggest that alive cells are required for an effective osmoporation-assisted encapsulation process; however, osmoporated biocapsules can efficiently protect and preserve labile compounds, independently of their activity. Overall, this study demonstrated that osmoporation using probiotic L. acidophilus is a simple, versatile, and efficient technique to encapsulate and deliver lipophilic fisetin for food applications. KEY POINTS : •Fisetin is efficiently encapsulated into L. acidophilus via osmoporation. •Fisetin bioaccessibility is improved by osmoporation into L. acidophilus. •Release mechanisms of osmoporation carriers are independent of the cell activity.