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Introduction Prosocial behavior has been widely associated with higher subjective wellbeing (SWB), yet little is known about whether blood donation, a distinct type of formal prosocial behavior, produces similar psychological benefits. Applied Self-Determination Theory, this study examined the relationship between blood donation and SWB, and explored whether satisfaction of basic psychological needs (BPN; autonomy, competence, and relatedness) explains this association. Understanding these psychological benefits may also provide insights for improving blood donor recruitment and retention. Methods A two-phase design was employed. Phase 1 was a cross-sectional survey comparing 601 whole blood donors (Donation group) with 1,043 non-donors, including participants recalling prosocial acts (Recall group, n = 517) and controls with no recall task (Non-recall group, n = 526). Phase 2 was a single-center randomized trial in which donors were allocated to receive follow-up questionnaires either with (Intervention group, n = 301) or without (Control group, n = 300) a gratitude reinforcement message highlighting their donation’s life-saving impact. SWB was assessed using measures of positive affect, negative affect, and life satisfaction. BPN satisfaction was measured using validated scales. ANCOVA models were used to estimate intervention effects while controlling for baseline levels. Results Cross-sectional analyses indicated that both blood donation and recalling prosocial behavior were positively associated with Time-1 SWB compared with the Non-recall group. Mediation analyses further showed that Time-1 BPN satisfaction significantly mediated the association between blood donation and Time-1 SWB. In the trial, ANCOVA models indicated that the gratitude reinforcement message significantly increased Time-2 BPN satisfaction but was not directly associated with Time-2 SWB. However, mediation analyses suggested an indirect pathway whereby the intervention was associated with higher Time-2 SWB through increased Time-2 BPN satisfaction, particularly competence satisfaction. Discussion This research represents a first step in understanding the relationship between blood donation and SWB. Findings suggest that blood donation may be associated with improved wellbeing partly through the satisfaction of BPN. Reinforcing donors’ perceived impact may strengthen these psychological benefits, although stronger feedback interventions may be required to produce measurable improvements in wellbeing. Future research should examine these effects longitudinally and across cultures. Clinical trial registration Clinicaltrials.gov , identifier NCT05213130.