Long non‐coding RNAs (lncRNAs) have been shown to play key roles in the pathogenesis of breast cancer (BC). The study was to explore the effect of long non‐coding RNA LINC00261/microRNA (miR)‐550a‐3p/serum deprivation response (SDPR) axis on the biological characteristics of BC stem cells (BCSCs). BC and adjacent normal tissues of patients were collected. LINC00261, miR‐550a‐3p and SDPR expression in BC tissues and cell lines and CD24 and CD44 expression in BC tissues was detected. CD44+/CD24−/low BCSCs were sorted. CD44+/CD24−/low MDA‐MB‐231 and MCF‐7 cells were screened and transfected with altered expression of LINC00261 or miR‐550a‐3p to explore their roles in cell viability, microsphere (MS) formation ability, migration and invasion of CD44+/CD24−/low BCSCs. The targeting relationships of LINC00261, miR‐550a‐3p and SDPR were detected. Reduced LINC00261, decreased SDPR and elevated miR‐550a‐3p exhibited in BC tissues of patients and cell lines. Elevated CD44+/CD24− were present in BC tissues. LINC00261 up‐regulated SDPR expression as a sponge of miR‐550a‐3p. Elevated LINC00261 suppressed BC cell viability, MS formation ability, migration and invasion of CD44+/CD24−/low BCSCs. Moreover, up‐regulated miR‐550a‐3p reversed the inhibitive effect of elevated LINC00261 on BCSCs, and reduced SDPR reversed the promotive effect of decreased miR‐550a‐3p on BCSCs. The study highlights that LINC00261 can adsorb miR‐550a‐3p to modulate SDPR, thus inhibiting the viability and MS formation of BC cells, reducing migration and invasion of CD44+/CD24−/low BCSCs, exerting a potential effect on therapy.