Náprstek laid the basis of the Japanese collection together with his wife, Josefa. The first items of Japanese provenance were acquired for the museum in 1863. Remarkable Japanese additions were acquired primarily from art dealers from Prague, Vienna, Hamburg, Leipzig and Bremen. In this earlier period, famous Czech artists and writers, for example Julius Zeyer, contributed to the collection. Among the friends of the museum were many travellers, like Josef Kořenský and Enrique Stanko Vráz, who frequently expanded the museum collections with didactic material gathered during their journeys. Václav Stejskal (1851–1934) was one of these travellers. He donated part of his collection in 1888 and passed the rest on to the museum after his death. Joe Hloucha (1881–1957), a Czech Japanophile and writer, travelled to Japan twice, in 1906 and 1926. A large part of Hloucha’s collection went to the Náprstek Museum between 1943 and 1955; including almost 8,000 items, it makes up around two fifths of the museum's Japanese collection (e.g. 4,000 prints, 530 photographs, 85 netsuke buttons, 77 sword guards, over 100 ceramic pieces, etc.).
The collection of Japanese prints is very extensive, numbering 7,000 items. Among the outstanding examples of Japanese woodblocks is a portrait of the actor Bando Hikosaburo by Tōshūsai Sharaku (active 1794–1795). The collection of prints includes black-and-white and hand-coloured photographs by Japanese photographers of the late 19th century, which Joe Hloucha bought during his first stay in Japan (525 pieces). Painting is represented in the Japanese collection by 700 works of varied quality. Possibly the oldest example of painting is the 14th century standing figure of Amida Buddha.
Another comprehensive part of the Japanese collection is the collection of Japanese ceramics, numbering around 1,600 pieces. Outstanding among them are about 100 pieces of Arita ware, originating from the porcelain kilns of the town of Arita on Kyushu Island, dating from the period from the late 17th to the early 19th century. The Náprstek Museum holds 170 pieces decorated using the cloissoné technique. About 400 lacquerware pieces make a small but pretty collection of artistic lacquer work, supplemented by the same amount of lacquer tableware. The collection of lacquers includes a set of Inrō containers, which carried personal seals and medicine, and netsuke buttons.
Another well represented Japanese craft is swordsmithing and sword decorating. The collection of swords holds about 220 swords of all types, from the oldest blades, dating from the 13th and 14th centuries, to souvenir goods from the 19th century. The collection also holds several complete sets of Samurai armour and over 200 individual parts, such as helmets, armoured sleeves, shin and thigh guards, facial armour and cuirasses.
Last but not least in the Japanese collection come a small set of traditional wooden toys, over 700 pieces of Japanese textiles, a comprehensive collection of Japanese fabrics (1,300) and 850 katagami stencils for dyeing fabrics, which illustrate above all the sophisticated and detailed contemporary ornamentation.