The transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues. Nitric oxide (NO) as a gaseous signal mediator shows a variety of important biological effects. In many instances, NO has been shown to exhibit its activities via a protein S-nitrosylation mechanism in order to regulate its protein functions. With functional assays via site-directed mutagenesis, we demonstrate herein that NO induces the S-nitrosylation of TRPV4 $Ca^{2+}$ channel on the $Cys^{853}$ residue, and the S-nitrosylation of $Cys^{853}$ reduced its channel sensitivity to 4-${alpha}$ phorbol 12,13-didecanoate and the interaction between TRPV4 and calmodulin. A patch clamp experiment and $Ca^{2+}$ image analysis show that the S-nitrosylation of $Cys^{853}$ modulates the TRPV4 channel as an inhibitor. Thus, our data suggest a novel regulatory mechanism of TRPV4 via NO-mediated S-nitrosylation on its $Cys^{853}$ residue.