The existence of SpoU-TrmD (SPOUT) RNA methyltransferase superfamily was first predicted by bioinformatics. SpoU is the previous name of TrmH, which catalyzes the 2'-O-methylation of ribose of G18 in tRNA; TrmD catalyzes the formation of N-1-methylguanosine at position 37 in tRNA. Although SpoU (TrmH) and TrmD were originally considered to be unrelated, the bioinformatics study suggested that they might share a common evolution origin and form a single superfamily. The common feature of SPOUT RNA methyltransferases is the formation of a deep trefoil knot in the catalytic domain. In the past decade, the SPOUT RNA methyltransferase superfamily has grown; furthermore, knowledge concerning the functions of their modified nucleosides in tRNA has also increased. Some enzymes are potential targets in the design of anti-bacterial drugs. In humans, defects in some genes may be related to carcinogenesis. In this review, recent findings on the tRNA methyltransferases with a SPOUT fold and their methylated nucleosides in tRNA, including classification of tRNA methyltransferases with a SPOUT fold; knot structures, domain arrangements, subunit structures and reaction mechanisms; tRNA recognition mechanisms, and functions of modified nucleosides synthesized by this superfamily, are summarized. Lastly, the future perspective for studies on tRNA modification enzymes are considered.