Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were studied as drug delivery vehicles for calcitriol, the active form of vitamin D3. In vitro effects of calcitriol encapsulated in PLGA nanoparticles were evaluated with respect to free calcitriol on human pancreatic cell lines, S2-013 and hTERT-HPNE, and the lung cancer cell line A549. Encapsulated calcitriol retained its biological activity, reducing the cell growth. Cytotoxicity assays demonstrated that encapsulation of calcitriol enhanced its inhibitory effect on cell growth at a concentration of 2.4 μM for the S2-013 cells (91%) and for A549 cells (70%) comparared to the free calcitriol results. At this concentration the inhibitory effect on nontumor cells (hTERT-HPNE) decreased to 65%. This study highlights the ability of PLGA nanoparticles to deliver vitamin D3 into cancer cells, with major effects regarding cancer cell cycle arrest and major changes in the cell morphological features.