Ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA) is a ubiquitous health beneficial phenolic acid. Although FA has shown a diversity of biological activities including anti-inflammatory, antihypercholesterolemic and anticancer bioactivities, studies revealing its adverse effects are accumulating. Recently, 3D-cultures are shown to exhibit uniquely biological behaviors different from that of 2D cultures. To understand whether the cytotoxicity of FA against the T24 cell line (a bladder cancer cell line) in 2D-culture could consistently retain similar bioactivity if cultured in the 3D-systems, we conducted this experiment with 2 mM FA. Much higher cytotoxicity was found for 3D- than 2D-culture, showing (2D vs. 3D): apoptotic rates, 64% and 76%; cell killing rates, 3.00 × 10(5) cells mmol(-1)·h(-1) and 2.63 × 10(6) cells mmol(-1)·h(-1), attaining a 8.77-fold. FA upregulated the activities at 72 h (2D vs. 3D in folds that of control): SOD, 1.73-folds (P < 0.05) versus 3.18 folds (P < 0.001); and catalase, 2.58 versus 1.33-folds. Comparing to the control (without FA), Bcl-2 was prominently downregulated while Bax, caspase-3 and cleaved caspase-9 were more upregulated in 3D-cultures (P < 0.05). Conclusively, different microenvironments could elicit different biological significance which in part can be ascribed to different mass transport rate.