Single photon sources based on semiconductor quantum dots offer distinct advantages for quantum information, including a scalable solid-state platform, ultrabrightness, and interconnectivity with matter qubits. A key prerequisite for their use in optical quantum computing and solid-state networks is a high level of efficiency and indistinguishability. Pulsed resonance fluorescence (RF) has been anticipated as the optimum condition for the deterministic generation of high-quality photons with vanishing effects of dephasing. Here, we generate pulsed RF single photons on demand from a single, microcavity-embedded quantum dot under s-shell excitation with 3-ps laser pulses. The π-pulse excited RF photons have less than 0.3% background contributions and a vanishing two-photon emission probability. Non-postselective Hong-Ou-Mandel interference between two successively emitted photons is observed with a visibility of 0.97(2), comparable to trapped atoms and ions. Two single photons are further used to implement a high-fidelity quantum controlled-NOT gate.Single photons have been proposed as promising quantum bits (qubits) for quantum communication [1], linear optical quantum computing [2, 3] and as messengers in quantum networks [4]. These proposals primarily rely upon a high degree of indistinguishability between individual photons to obtain the Hong-Ou-Mandel (HOM) type interference [5] which is at the heart of photonic controlled logic gates and photon-interference-mediated quantum networking [1][2][3][4].Among different types of single-photon emitters [6, 7], quantum dots (QDs) are attractive solid-state devices since they can be embedded in high-quality nanostructure cavities and waveguides to generate ultra-bright sources of single and entangled photons [7][8][9][10]. QDs also provide a light-matter interface [11][12][13] and can in principle be scaled to large quantum networks [14]. Two-photon HOM interference experiments using photons from a single QD [5,15,17], as well as from independent sources [18,19], have not only demonstrated the potential of QDs as single-photon sources, but also revealed the level of dephasing arising from incoherent excitation. The method of incoherent pumping (via above band-gap or p-shell excitation) typically causes reduced photon coherence times due to homogeneous broadening of the excited state [5] and uncontrolled emission time jitter from the nonradiative high-level to s-shell relaxation [6], leading to a decrease of photon indistinguishability.To eliminate these dephasings, an increasing effort has been devoted to s-shell resonant optical excitation of QDs. The Mollow triplet spectra and photon correlations of the resonance fluorescence (RF) have been measured [1][2][3]21]. Under continuous-wave (CW) laser excitation, a high degree of indistinguishability for continuously generated RF photons has been demonstrated through post-selective HOM interference [25]. However, in the CW regime, as the emission time of the RF photons is uncontrolled, the HOM interference relies on th...