This paper describes the experimental investigation of orthogonal time-frequency space (OTFS) modulation using a 28 GHz multi-user distributed multiple-input multiple-output (D-MIMO) testbed in over-the-air (OTA) and mobility environments to enhance cell throughput in a millimeter-wave band. We build the D-MIMO testbed having newly developed OTFS modulator and demodulator, and measured OTFS signals and orthogonal frequency-division multiplexing (OFDM) signals with up to four user simultaneous connections on an actual office floor. Additionally, the Doppler effect on OTFS signals is mathematically analyzed and is confirmed in the measurements. OTFS indicates higher robustness in time-variant channels than OFDM. The error vector magnitude (EVM) and system throughput of OTFS are −22 dB and 1.9 Gbps with 100 MHz signal bandwidth, respectively. To our knowledge, this is a first paper describing the OTA measurements of EVM, throughput, and spectral efficiency using OTFS modulation on the 28 GHz coherent beamforming system.