High-resolution X-ray spectroscopy with Hitomi was expected to resolve the origin of the faint unidentified E ≈ 3.5 keV emission line reported in several low-resolution studies of various massive systems, such as galaxies and clusters, including the Perseus cluster. We have analyzed the Hitomi first-light observation of the Perseus cluster. The emission line expected for Perseus based on the XMM-Newton signal from the large cluster sample under the dark matter decay scenario is too faint to be detectable in the Hitomi data. However, the previously reported 3.5 keV flux from Perseus was anomalously high compared to the sample-based prediction. We find no unidentified line at the reported high flux level. Taking into account the XMM measurement uncertainties for this region, the inconsistency with Hitomi is at a 99% significance for a broad dark matter line and at 99.7% for a narrow line from the gas. We do not find anomalously high fluxes of the nearby faint K line or the Ar satellite line that were proposed as explanations for the earlier 3.5 keV detections. We do find a hint of a broad excess near the energies of high-n transitions of S XVI (E ; 3.44 keV rest-frame)—a possible signature of charge exchange in the molecular nebula and another proposed explanation for the unidentified line. While its energy is consistent with XMM pn detections, it is unlikely to explain the MOS signal. A confirmation of this interesting feature has to wait for a more sensitive observation with a future calorimeter experiment.