Dietary intake of long chain omega-3 polyunsaturated fatty acids (n-3 PUFA), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has been associated with psychological well-being, but little is known about the n-3 PUFA intake of homeless youth. This study determined the association between depression and anxiety symptoms and n-3 PUFA intake and red blood cell (RBC) status in homeless youth. 114 homeless youth aged 18-24 years were recruited from a drop-in center. n-3 PUFA dietary intake was assessed using a Food Frequency Questionnaire (FFQ), and RBC status was determined by gas chromatography. Linear regression models were used to determine the relationship between psychological well-being and n-3 PUFA intake and status. The mean intakes of EPA and DHA for all participants (0.06 ± 0.13 g/day and 0.11± 0.24 g/d,) were well below recommended levels, and mean RBC EPA + DHA (omega-3 index) in the cohort (2.42%) was lower than reported for healthy, housed adolescents and those with clinical depression. There was no association of n-3 PUFA intake and RBC status with either depression or anxiety. However, the relationships of depression with dietary EPA (p = 0.017), and DHA (p=0.008) as well as RBC DHA (p = 0.007) and omega 3-index (p= 0.009), were significantly moderated by sex even after adjusting for confounders. Specifically, among females, as the intake and status of these n-3 PUFAs decreased, depression increased. Our findings show poor dietary intake and low RBC status of n-3 PUFA among homeless youth, which is associated with depressive symptoms among females.