Nitrogen (N) is an essential major nutrient for food crops. Although ammonium (NH4+) is the primary N source of rice (Oryza sativa), nitrate (NO3-) can also be absorbed and utilized. Rice responds to NO3- application by altering its root morphology, such as root elongation. Strigolactones (SLs) are important modulators of root length. However, the roles of SLs and their downstream genes in NO3--induced root elongation remain unclear. Here, the levels of total N and SL (4-deoxyorobanchol) and the responses of seminal root (SR) lengths to NH4+ and NO3- were investigated in rice plants. NO3- promoted SR elongation, possibly due to short-term signal perception and long-term nutrient function. Compared with NH4+ conditions, higher SL signalling/levels and less D53 protein were recorded in roots of NO3--treated rice plants. In contrast to wild-type plants, SR lengths of d mutants were less responsive to NO3- conditions, and application of rac-GR24 (SL analogue) restored SR length in d10 (SL biosynthesis mutant) but not in d3, d14, and d53 (SL-responsive mutants), suggesting that higher SL signalling/levels participate in NO3--induced root elongation. D53 interacted with SPL17 and inhibited SPL17-mediated transactivation from the PIN1b promoter. Mutation of SPL14/17 and PIN1b caused insensitivity of the root elongation response to NO3- and rac-GR24 applications. Therefore, we conclude that perception of SLs by D14 leads to degradation of D53 via the proteasome system, which releases the suppression of SPL14/17-modulated transcription of PIN1b, resulting in root elongation under NO3- supply.