In mass spectrometry (MS), a major loss of ions can readily occur during their transfer from atmospheric pressure to a lower pressure, which limits performance. Here, we report an ion funnel that can be used to effectively focus ions at ambient pressure (∼777 Torr) to significantly enhance performance in electrospray ionization (ESI) MS. For seven singly charged test ions (m/z 124-1131), the ambient pressure ion funnel (APIF) is demonstrated to improve ion abundances, sensitivity, and detection limits by up to factors of ∼17, ∼16, and ∼3, respectively, compared to the operation of conventional ESI-MS. Simulated ion trajectories were used to rationalize the enhanced performance of the APIF, which is attributed primarily to using a relatively high RF field amplitude to radially confine ions, a high DC field, and a wide exit ring electrode. The effective focusing of ions at ambient pressures should be beneficial in the future for improving the performance of (i) additional methods that ionize molecules at atmospheric pressure, (ii) ambient pressure ion mobility-based instruments, and (iii) high flow rate liquid chromatography mass spectrometry platforms.