Fine roots, an important component of the forest ecosystem in terms of carbon and nutrient cycles, are highly heterogenous in structure and function. Previous studies revealed that fine roots of different branch orders differed in morphology; however, few studies have focused on the functions of the different orders of roots in terms of ecophysiology. We here aimed to (1) measure root respiration rate along the branch orders using gas-phase O2 electrodes as O2 consumption in samples of Larix gmelinii and Fraxinus mandshurica and (2) examine the relationship between fine-root respiration and morphology (diameter, root length, specific root length), anatomy (cortex, stele), and tissue N concentration in the 2 species. With increasing root order (from first- to fifth-order roots), fine-root diameter and length and stele diameter increased, but specific root length (SRL), tissue N and root respiration rate decreased, with marked differences between both tree species (P < 0.05). First-order roots with an intact cortex, the smallest diameter and highest SRL and tissue N concentration had the highest respiration rate: 17.57 and 18.80 nmolO2·g–1·s–1 (L. gmelinii and F. mandshurica, respectively). In contrast, fifth-order roots showed the highest diameter and the lowest SRL and N concentration with no cortex, and thus had the lowest respiration rate. The difference in respiration rate between the first- and fifth-order roots was 148% (L. gmelinii) and 124% (F. mandshurica), respectively. In addition, 96% (L. gmelinii) and 89% (F. mandshurica) of fine-root respiration rate was related to tissue N concentration. Fine-root physiological functions may be closely related to their morphology, anatomy and tissue N concentration. First-order roots with high respiration rate represent absorptive roots, and fourth- and fifth-order roots may have limited uptake capacity and function in transportation and storage.