Nitric oxide (NO) is a key molecule in blood pressure regulation, neuromodulation, and biodefense. Since it is an unstable gas under ambient conditions, various NO donors have been developed and employed for biological studies as an alternative to direct NO application. However, many of them release NO via spontaneous decomposition, so that it is difficult to control the NO release. Therefore, NO donors from which NO release can be temporally and spatially well-controlled by means of photoexcitation offer considerable advantages. Various NO donors from which NO release is activated by photoirradiation have been reported. However, the maximum absorption of these NO donors is generally limited to the UV or short-wavelength visible light range, which does not adequately penetrate living tissues. To overcome this limitation, NO donors working via two-photon excitation (TPE) were developed by Ford and Prasad, based on NO release from Fe-nitrosyl complex. We have also developed TPE-type NO donors based on our photo-controllable 2,6-dimethylnitrobenzene derivatives, and investigated their NO-releasing properties. Here, we present an overview of photocontrollable NO donors and discuss their properties in relation to biological applications.