Little Sister may refer to:

• Birth order

• The Little Sister, a 1949 novel by Raymond Chandler
• Kana: Little Sister, a 1999 Japanese visual novel

• The Little Sister (1911 film), a short
• The Little Sister (1914 film), a short starring Tom Mix
• Little Sister (1921 film), a 1921 Italian silent film
• The Little Sister (1986 film), featuring Peter Gerety
• Little Sister (1992 film), a comedy starring Jonathan Silverman and Alyssa Milano
• Little Sister (1995 film), a Dutch film
• Little Sister (1999 film), a Finnish film
• Little Sister (2010 film), directed and written by Richard Bowen
• Little Sister (2016 film), directed and written by Zach Clark
• "The Little Sister" (Roseanne), a 1989 episode of the TV sitcom Roseanne
• "Little Sister" (Golden Girls episode)

• Little Sister (band), Sly & the Family Stone's background vocalists
• "Little Sister" (Elvis Presley song), a song released by Elvis Presley in 1961, and later covered by Ry Cooder and by Dwight Yoakam
• "Little Sister" (Queens of the Stone Age song)
• "Little Sister", a song by Jewel from Pieces of You
• "Little Sister", a song by the Runaways from Waitin' for the Night
• "Little Sister", a song by Lou Reed from the Get Crazy soundtrack
• "Little Sister", a song by Nico from Chelsea Girl
• "Little Sister", a song by Cheap Trick from Standing on the Edge
• "Bonnie Jean (Little Sister)", by David Lynn Jones
• "Little Sister", a song by Trixie Mattel from One Stone

• Little Sisters of the Abandoned Elderly, founded in Spain, a Roman Catholic religious congregation
• Little Sisters of the Assumption, a Roman Catholic religious institute
• Little Sisters of Jesus, a Roman Catholic congregation
  • Little Sister Magdeleine of Jesus (1898–1989), founder of the Little Sisters of Jesus
• Little Sisters of the Lamb, a branch of a Roman Catholic religious institute, shaped both by Dominican and by Franciscan spirituality
• Little Sisters of the Poor, founded in France, a Roman Catholic religious institute for women
• Little Sisters of the Sacred Heart, a religious congregation in Montpellier

• Little Sisters, an informal name for a group of some of the smaller members of the British Virgin Islands
• Little Sister (BioShock), fictional young girls in BioShock and BioShock 2
• Little Sister's Book and Art Emporium, a bookstore in Vancouver, Canada
  • Little Sisters Book and Art Emporium v Canada (Minister of Justice), a court case regarding freedom of speech involving the bookstore

• Nation's Little Sister, an informal title in the South Korean entertainment industry
• "Baby Sister", a 1985 song by La Toya Jackson
• LittleSis, an accountability project of the Public Accountability Initiative
• All pages with titles beginning with Little Sister
• All pages with titles containing Little Sister
• Lillasyster (Swedish: little sister), a Swedish rock band
• Petite soeur (disambiguation) (French: little sister)
• Mei Mei (disambiguation) (Chinese: 妹妹; lit. 'little younger sister')