This study aimed at assessing the effects of space allowance on microclimate and pig stress during transportation in a mechanically-ventilated vehicle. On each journey (6), 114 pigs were randomly assigned to different space allowances (0.46, 0.49 or 0.59 m2/pig) tested in the near-front, middle and near-rear compartments and on the top, middle and bottom decks. In each compartment, ambient conditions (e.g., T°C and RH%) were monitored. Behaviour was recorded during lairage. A total of 162 pigs were selected for the analysis of blood hematocrit proportion and lactate, and creatine kinase (CK) concentrations, and the evaluation of meat quality in the loin and ham muscles. The bottom deck was more humid before and during transport (P ≤ 0.05) and pigs from this location stood and drank more in lairage (P < 0.001). Pigs transported on the top deck had greater blood lactate and CK concentrations (P < 0.05) than those transported on the bottom deck, and when transported at 0.49 m2/pig produced paler loins (P < 0.05) than those transported at 0.46 m2/pig. The within-trailer location had a greater impact on the microclimate, collected animal welfare measures, and meat quality of pigs transported in an actively ventilated trailer than space allowance.