The protein N-terminal methyltransferase 1 (NTMT1) is implicated in neurogenesis, retinoblastoma, and cervical cancer. However, its pharmacological potentials have not been elucidated due to the lack of drug-like inhibitors. Here, we report the discovery of the first NTMT1 in vivo chemical probe GD433 by structure-guided optimization of our previously reported lead compound venglustat. GD433 (IC50 = 27 ± 1.1 nM) displays improved potency and selectivity than venglustat across biochemical, biophysical, and cellular assays. GD433 also displays good oral bioavailability and can serve as an in vivo chemical probe to dissect the pharmacological roles of Nα methylation. In addition, we also identified a close analogue (YD2160) that is inactive against NTMT1. The active inhibitor and negative control will serve as valuable tools to examine the physiological and pharmacological functions of NTMT1 catalytic activity.